Calicheamicin-antibody-drug conjugates and methods of use

ABSTRACT

The invention relates generally to a calicheamicin molecule activated with a leaving group. The invention further relates generally to an antibody-drug conjugate comprising an antibody directly conjugated by a disulfide to one or more calicheamicin molecules.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority benefit of U.S. provisional applicationSer. No. 62/243,967 filed on Oct. 20, 2015, which is incorporated hereinin its entirety.

FIELD OF THE INVENTION

The field of the invention relates generally to an antibody-drugconjugate comprising an antibody directly conjugated to one or morecalicheamicin molecules.

BACKGROUND

Calicheamicin and calicheamicin derivatives refer to a family ofantibacterial and antitumor agents, as described, for instance, in U.S.Pat. No. 4,970,198 (which is incorporated herein in its entirety).Calicheamicin derivatives within the scope of the disclosure include,without limitation, dihydro derivatives as described in U.S. Pat. No.5,037,651 and N-acylated derivatives as described in U.S. Pat. No.5,079,233 (both of which are incorporated in their entirety herein). Asused herein, a calicheamicin derivative refers to calicheamicin that hasbeen substituted at one or more positions to obtain a differentcompound.

The calicheamicin family of antibiotics, and derivatives and analogsthereof, are capable of producing double-stranded DNA breaks atsub-picomolar concentrations (Hinman et al., (1993) Cancer Research53:3336-3342; Angew Chem. Intl. Ed. Engl. (1994) 33:183-186; Lode etal., (1998) Cancer Research 58:2925-2928). Calicheamicin comprises awarhead comprising an enediyne ring structure (a ring comprising adouble bond flanked by triple bonds) and a methyl trisulfide (i.e.,—S—S—S—CH₃) group. It is believed that the warhead is activated byreduction of a disulfide bond, and that the activated warhead functionsby causing breaks in double-stranded DNA. A mechanism of action wasproposed by Bouchard, H., et al., Ab-drug conjugates-A new wave ofcancer drugs, Bioorganic & Medicinal Chemistry Letters 24 (2014)5357-5363 where the enediyne ring is activated by reductive cleavage ofthe disulfide bond by the steps: (i) formation of acalicheamicin=CHCH₂SH moiety by nucleophilic attack of the methyltrisulfide moiety and cleavage of CH₃—S—S—, (ii) formation of a fused2,5-dihydrothiophene ring from calicheamicin=CHCH₂SH, and (iii)formation of a fused benzene free di-radical from the enediyne.Activated calicheamicin then cleaves double stranded DNA.

Calicheamicin has intracellular sites of action, but, in some instances,does not effectively cross the plasma membrane. Therefore, cellularuptake of these agents through antibody-mediated internalization may, insome embodiments, greatly enhance cytotoxic effect. It is known thatcalicheamicin-linker-antibody conjugates provide for the specificity andeffective plasma membrane permeability (internalization) of the antibodyin combination with the cytotoxic potency of calicheamicin. Therefore,cellular uptake of calicheamicin may, in some aspects, greatly enhanceits cytotoxic effect. Methods of forming calicheamicin-linker-antibodydrug conjugates are known and described, for example, in U.S. Pat. Nos.5,877,296, 5,773,001, 5,712,374, 5,714,586, 5,739,116 and 5,767,285(each of which is incorporated by reference herein).

Antibody-drug conjugates, comprising an antibody-linker-drug conjugate,are attractive targeted chemo-therapeutic molecules, as they combineideal properties of both antibodies and cytotoxic drugs by targetingpotent cytotoxic drugs to the antigen-expressing tumor cells, therebyenhancing their anti-tumor activity. Successful antibody-drug conjugatedevelopment for a given target antigen depends on optimization ofantibody selection, linker stability, cytotoxic drug potency and mode oflinker-drug conjugation to the antibody. More particularly, effectiveantibody-drug conjugates are characterized by at least one or more ofthe following: (i) an antibody-drug conjugate formation method whereinthe antibody retains sufficient specificity to target antigens andwherein the drug efficacy is maintained; (ii) antibody-drug conjugatestability sufficient to limit drug release in the blood and concomitantdamage to non-targeted cells; (iii) sufficient cell membrane transportefficiency (endocytosis) to achieve a therapeutic intracellularantibody-drug conjugate concentration; (iv) sufficient intracellulardrug release from the antibody-drug conjugate sufficient to achieve atherapeutic drug concentration; and (v) drug cytotoxicity in nanomolaror sub-nanomolar amounts.

Conventional means of attaching, i.e., covalent bonding of a drug moietyto an antibody via a linker, generally leads to a heterogeneous mixtureof molecules where the drug moieties are attached at a number of siteson the antibody. For example, cytotoxic drugs have typically beenconjugated to antibodies through the often-numerous lysine residues ofan antibody, generating a heterogeneous antibody-drug conjugate mixture.Depending on reaction conditions, the heterogeneous mixture typicallycontains a distribution of antibodies with from 0 to about 8, or more,attached drug moieties. In addition, within each subgroup of conjugateswith a particular integer ratio of drug moieties to a single antibody,there is a potentially heterogeneous mixture where the drug moiety isattached at various sites on the antibody. Analytical and preparativemethods are inadequate to separate and characterize the antibody-drugconjugate species molecules within the heterogeneous mixture resultingfrom a conjugation reaction. Antibodies are large, complex andstructurally diverse biomolecules, often with many reactive functionalgroups. Antibody reactivity with linker reagents and drug-linkerintermediates are dependent on factors such as pH, concentration, saltconcentration, and co-solvents. Furthermore, the multistep conjugationprocess may be nonreproducible due to difficulties in controlling thereaction conditions and characterizing reactants and intermediates.

Antibody-drug conjugates are typically formed by conjugating one or moreantibody cysteine thiol groups to one or more linker moieties bound to adrug thereby forming an antibody-linker-drug complex. Cysteine thiolsare reactive at neutral pH, unlike most amines which are protonated andless nucleophilic near pH 7. Since free thiol (RSH, sulfhydryl) groupsare relatively reactive, proteins with cysteine residues often exist intheir oxidized form as disulfide-linked oligomers or have internallybridged disulfide groups. Antibody cysteine thiol groups are generallymore reactive, i.e. more nucleophilic, towards electrophilic conjugationreagents than antibody amine or hydroxyl groups. Engineering in cysteinethiol groups by the mutation of various amino acid residues of a proteinto cysteine amino acids is potentially problematic, particularly in thecase of unpaired (free Cys) residues or those which are relativelyaccessible for reaction or oxidation. In concentrated solutions of theprotein, whether in the periplasm of E. coli, culture supernatants, orpartially or completely purified protein, unpaired Cys residues on thesurface of the protein can pair and oxidize to form intermoleculardisulfides, and hence protein dimers or multimers. Disulfide dimerformation renders the new Cys unreactive for conjugation to a drug,ligand, or other label. Furthermore, if the protein oxidatively forms anintramolecular disulfide bond between the newly engineered Cys and anexisting Cys residue, both Cys groups are unavailable for active siteparticipation and interactions. Furthermore, the protein may be renderedinactive or non-specific, by misfolding or loss of tertiary structure(Zhang et al. (2002) Anal. Biochem. 311:1-9).

Improved antibody-drug conjugates, THIOMAB™, have been developed thatprovide for site-specific conjugation of a drug to an antibody throughcysteine substitutions at sites where the engineered cysteines areavailable for conjugation but do not perturb immunoglobulin folding andassembly or alter antigen binding and effector functions (Junutula, etal., 2008b Nature Biotech., 26(8):925-932; Dornan et al. (2009) Blood114(13):2721-2729; U.S. Pat. No. 7,521,541; U.S. Pat. No. 7,723,485;WO2009/052249). These THIOMAB™ antibodies can then be conjugated tocytotoxic drugs through the engineered cysteine thiol groups to obtainTHIOMAB™ drug conjugates (TDC) with uniform stoichiometry (e.g., up to 2drugs per antibody in an antibody that has a single engineered cysteinesite). Studies with multiple antibodies against different antigens haveshown that TDCs are as efficacious as conventional antibody-drugconjugate in xenograft models and are tolerated at higher doses inrelevant preclinical models. THIOMAB™ antibodies have been engineeredfor drug attachment at different locations of the antibody (e.g.,specific amino acid positions (i.e., sites) within the light chain-Fab,heavy chain-Fab and heavy chain-Fc). The in vitro and in vivo stability,efficacy and PK properties of THIOMAB™ antibodies provide a uniqueadvantage over conventional antibody-drug conjugates due to theirhomogeneity and site-specific conjugation to cytotoxic drugs.

There are still other limitations or challenges to the preparation anduse of antibody-drug conjugates, and in particularantibody-calicheamicin derivative conjugates. For example, some linkersmay be labile in the blood stream, thereby releasing unacceptableamounts of the drug prior to internalization in a target cell. Otherlinkers may provide stability in the bloodstream, but intracellularrelease effectiveness may be negatively impacted. Linkers that providefor desired intracellular release typically have poor stability in thebloodstream. Alternatively stated, bloodstream stability andintracellular release are typically inversely related. Second, instandard conjugation processes, the amount of calicheamicin loaded onthe carrier protein (the drug loading), the amount of aggregate that isformed in the conjugation reaction, and the yield of final purifiedconjugate that can be obtained are interrelated. For example, aggregateformation is generally positively correlated to the number ofequivalents of calicheamicin and derivatives thereof conjugated to thecarrier-antibody. Because drug potency and efficacy increases withcalicheamicin content, it is desirable to maximize calicheamicin loadingon an antibody carrier while retaining the affinity of the antibody.However, under high drug loading, formed aggregates must be removed fortherapeutic applications. As a result, drug loading-mediated aggregateformation decreases antibody-drug conjugate yield and can rendersprocess scale-up difficult. For example, prior art conjugation methodsusing linkers have been found to require a compromise between higherdrug loading and antibody-drug conjugate yield, by limiting the amountof calicheamicin that is added to the conjugation reaction.

Accordingly, there is a continuing need for improved efficaciouscalicheamicin-antibody conjugates that provide for optimized safety andefficacy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a plot of in vivo tumor volume change over time in aHCC1569X2 xenograph model in SCID Beige mice after IV dosing with ThioHu anti-Ly6E LC K149C-p-nitro-PDS-Calicheamicin at doses of 0.3 mg/kg, 1mg/kg, 3 mg/kg, 6 mg/kg and 10 mg/kg and with a Thio Hu anti-CD22 LCK149C-p-nitro-PDS-Calicheamicin control dosed IV at 3 mg/kg

FIG. 2 shows a plot of in vivo tumor volume change over time in aWSU-DLCL2 xenograph model in CB-17 Fox Chase SCID mice after IV dosingwith Thio Hu anti-CD22 10F4v3 LC K149C-p-nitro-PDS-Calicheamicin atdoses of 0.3 mg/kg, 1 mg/kg, 3 mg/kg, 6 mg/kg and 10 mg/kg and with aThio Hu anti-Ly6E 9B12v12 LC K149C-p-nitro-PDS-Calicheamicin controldosed IV at 3 mg/kg

FIG. 3A shows a plot of IC₅₀ potency versus log concentration againstCD22 positive Burkitt's human lymphoma cells (BJAB) for targeted Thio HuAnti-CD22 10F4v3 LC K149C-Calicheamicin IC50 as compared to non-targetedcontrol Thio Hu Anti-Ly6E 9B12.v12 LC K149C-Calicheamicin. The targetedpotency is >1500-fold greater than non-targeted potency.

FIG. 3B shows a plot of IC₅₀ potency versus log concentration againstCD22 positive WSU-DLCL2 human diffuse large B-cell lymphoma-derived cellline for targeted Thio Hu Anti-Ly6E 10F4v3 LC K149C-Calicheamicin IC₅₀potency as compared to non-targeted control Thio Hu Anti-Ly6E 9B12.v12LC K149C-Calicheamicin. The targeted potency is >2000-fold greater thannon-targeted potency.

FIG. 3C shows a plot of IC₅₀ potency versus log concentration againstJurkat cells for Thio Hu Anti-Ly6E 10F4v3 LC K149C-Calicheamicin andThio Hu Anti-Ly6E 9B12.v12 LC K149C-Calicheamicin IC₅₀ potency versuslog concentration.

SUMMARY

In one aspect of the present disclosure drug intermediates of Formula Iand of Formula II are provided:

In such aspects, R is selected from H, —C(O)R¹, —C(O)NR¹R², —S(O)₂R¹,and —S(O)2NR²R¹; R¹ and R² are independently selected from C₁-C₆ alkyland C₆-C₂₀ aryl; R³ is selected from NO₂, Cl, F, CN, CO₂H, and Br; and qis 0, 1, or 2.

In another aspect of the present disclosure, an antibody-drug conjugateof Formula III, or a pharmaceutically acceptable salt thereof, isprovided:

In such aspects, R is selected from H, —C(O)R¹, —C(O)NR¹R², —S(O)₂R¹,and —S(O)₂NR²R¹, and R¹ and R² are independently selected from C₁-C₆alkyl and C₆-C₂₀ aryl. The designator p is an integer from 1 to 8. Ab isan antibody which binds to one or more tumor-associated antigens orcell-surface receptors selected from (1)-(53), as listed herein: (1)BMPR1B (bone morphogenetic protein receptor-type IB); (2) E16 (LAT1,SLC7A5); (3) STEAP1 (six transmembrane epithelial antigen of prostate);(4) MUC16 (0772P, CA125); (5) MPF (MPF, MSLN, SMR, megakaryocytepotentiating factor, mesothelin); (6) Napi2b (NAPI-3B, NPTIIb, SLC34A2,solute carrier family 34 (sodium phosphate), member 2, type IIsodium-dependent phosphate transporter 3b); (7) Sema 5b (FLJ10372,KIAA1445, Mm.42015, SEMASB, SEMAG, Semaphorin 5b Hlog, sema domain,seven thrombospondin repeats (type 1 and type 1-like), transmembranedomain (TM) and short cytoplasmic domain, (semaphorin) 5B); (8) PSCA hlg(2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12, RIKEN cDNA2700050C12 gene); (9) ETBR (Endothelin type B receptor); (10) MSG783(RNF124, hypothetical protein F1120315); (11) STEAP2 (HGNC_8639, IPCA-1,PCANAP1, STAMP1, STEAP2, STMP, prostate cancer associated gene 1,prostate cancer associated protein 1, six transmembrane epithelialantigen of prostate 2, six transmembrane prostate protein); (12) TrpM4(BR22450, F1120041, TRPM4, TRPM4B, transient receptor potential cationchannel, subfamily M, member 4); (13) CRIPTO (CR, CR1, CRGF, CRIPTO,TDGF1, teratocarcinoma-derived growth factor); (14) CD21 (CR2(Complement receptor 2) or C3DR (C3d/Epstein Barr virus receptor) or Hs73792); (15) CD79b (CD79B, CD79β, IGb (immunoglobulin-associated beta),B29); (16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containingphosphatase anchor protein 1a), SPAP1B, SPAP1C); (17) HER2; (18) NCA;(19) MDP; (20) IL20Rα; (21) Brevican; (22) EphB2R; (23) ASLG659; (24)PSCA; (25) GEDA; (26) BAFF-R (B cell-activating factor receptor, BLySreceptor 3, BR3); (27) CD22 (B-cell receptor CD22-B isoform); (28) CD79a(CD79A, CD79a, immunoglobulin-associated alpha); (29) CXCR5 (Burkitt'slymphoma receptor 1); (30) HLA-DOB (Beta subunit of MHC class IImolecule (Ia antigen)); (31) P2X5 (Purinergic receptor P2X ligand-gatedion channel 5); (32) CD72 (B-cell differentiation antigen CD72, Lyb-2);(33) LY64 (Lymphocyte antigen 64 (RP105), type I membrane protein of theleucine rich repeat (LRR) family); (34) FcRH1 (Fc receptor-like protein1); (35) FcRH5 (IRTA2, Immunoglobulin superfamily receptor translocationassociated 2); (36) TENB2 (putative transmembrane proteoglycan); (37)PMEL17 (silver homolog; SILV; D12S53E; PMEL17; SI; SIL); (38) TMEFF1(transmembrane protein with EGF-like and two follistatin-like domainsTomoregulin-1); (39) GDNF-Ra1 (GDNF family receptor alpha 1; GFRA1;GDNFR; GDNFRA; RETL1; TRNR1; RET1L; GDNFR-alpha1; GFR-ALPHA-1); (40)Ly6E (lymphocyte antigen 6 complex, locus E; Ly67, RIG-E, SCA-2, TSA-1);(41) TMEM46 (shisa homolog 2 (Xenopus laevis); SHISA2); (42) Ly6G6D(lymphocyte antigen 6 complex, locus G6D; Ly6-D, MEGT1); (43) LGR5(leucine-rich repeat-containing G protein-coupled receptor 5; GPR49,GPR67); (44) RET (ret proto-oncogene; MEN2A; HSCR1; MEN2B; MTC1; PTC;CDHF12; Hs.168114; RET51; RET-ELE1); (45) LY6K (lymphocyte antigen 6complex, locus K; LY6K; HSJ001348; FLJ35226); (46) GPR19 (Gprotein-coupled receptor 19; Mm.4787); (47) GPR54 (KISS1 receptor;KISS1R; GPR54; HOT7T175; AXOR12); (48) ASPHD1 (aspartatebeta-hydroxylase domain containing 1; LOC253982); (49) Tyrosinase (TYR;OCAIA; OCA1A; tyrosinase; SHEP3); (50) TMEM118 (ring finger protein,transmembrane 2; RNFT2; FLJ14627); (51) GPR172A (G protein-coupledreceptor 172A; GPCR41; FLJ11856; D15Ertd747e); (52) CD33; and (53)CLL-1.

In some other aspects of the disclosure, pharmaceutical compositionscomprising an antibody-drug conjugate of the present disclosure isprovided, the pharmaceutical composition further comprising at least oneof a diluent, a carrier, and an excipient.

In other aspects, a pharmaceutical composition comprising anantibody-drug conjugate of the present disclosure and at least one of adiluent, a carrier, and an excipient is provided for administration to apatient for the treatment of cancer.

In still other aspects, antibody-drug conjugates of the presentdisclosure are provided for use in the manufacture of a medicament forthe treatment of cancer in an animal.

In yet other aspects, methods for treating cancer with the antibody-drugconjugates of the present disclosure are provided.

In other aspects, a method of making antibody-drug conjugates of thepresent disclosure is provided wherein the method comprises reacting anantibody which binds with one or more tumor-associated antigens orcell-surface receptors selected from (1)-(53) as described elsewhereherein with a drug-leaving group intermediate composition of formula Ior formula II as described elsewhere herein.

In other aspects, an article of manufacture is provided, the articlecomprising: an antibody-drug conjugate of the present disclosure and atleast one of a diluent, a carrier, and an excipient; a container; and apackage insert or label indicating that the pharmaceutical compositioncan be used to treat cancer.

DETAILED DESCRIPTION

Reference will now be made in detail to certain embodiments of theinvention, examples of which are illustrated in the accompanyingstructures and formulas. While the invention will be described inconjunction with the enumerated embodiments, it will be understood thatthey are not intended to limit the invention to those embodiments. Onthe contrary, the invention is intended to cover all alternatives,modifications, and equivalents, which may be included within the scopeof the present invention as defined by the claims.

One skilled in the art will recognize many methods and materials similaror equivalent to those described herein, which could be used in thepractice of the present invention. The present invention is in no waylimited to the methods and materials described.

The present disclosure is generally directed to antibody-drug conjugatescomprising an antibody directly conjugated to one or more calicheamicinderivatives, in the absence of a linker or linking moiety. The presentdisclosure is further directed to calicheamicin derivative intermediatecompositions comprising a leaving group. Such intermediate compositionsare suitable substrates for formation of antibody-drug conjugateswherein an antibody is covalently bound directly to calicheamicinderivative, after loss of the leaving group and in the absence of aconventional linker or linking moiety. The present disclosure is furtherdirected to use of such an antibody-calicheamicin conjugate in thetreatment of an illness, in particular cancer. As used herein, unlessotherwise specified, calicheamicin refers to the calicheamicinderivative compounds encompassed by the present disclosure.

In this regard it is to be noted that prior art methods for formingantibody-calicheamicin and antibody-calicheamicin derivative conjugates,such as those disclosed in U.S. 55,877,296 and 5,773,001, result in alarge percentage of conjugate aggregates, rendering scale-up impracticaland presenting purification problems (see U.S. publication no.2007/0213511 A1, which is incorporated herein by reference, at, forexample, paragraphs [0008] and [0009]). It is further known that themethods disclosed in U.S. Pat. No. 5,714,586 and U.S. Pat. No. 5,712,374produce antibody-drug conjugates having from 50% to 60% of an undesiredlow conjugated fraction (see, e.g., U.S. publication no. 2007/0213511 A1at paragraph [0010]). Furthermore, problematically, linkers may beinstable in the bloodstream, thereby resulting significant drug releaseprior to internalization. Further, the use of suchcalicheamicin-linker-antibody conjugates may be limited by thecapabilities of known conjugation processes, which typically result inthe formation aggregates, particularly when the drug loading perantibody molecule is increased.

Based on experimental evidence to-date, it has been discovered that thedirect conjugation, by means of a disulfide covalent bond between anantibody and a calicheamicin derivative, in the absence of a linker,provides for improved antibody-calicheamicin conjugates, characterizedby reproducible calicheamicin drug loading per antibody (DAR), reducedaggregate formation, improved bloodstream stability and improvedintracellular release. Accordingly, it is believed that directconjugation of an antibody to calicheamicin by a disulfide bondeffectively decouples bloodstream stability and intracellular releasesuch that both improved bloodstream stability and improved intracellularrelease are enabled.

DEFINITIONS

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs, and are consistent with:Singleton et al. (1994) Dictionary of Microbiology and MolecularBiology, 2nd Ed., J. Wiley & Sons, New York, N.Y.; and Janeway, C.,Travers, P., Walport, M., Shlomchik (2001) Immunobiology, 5th Ed.,Garland Publishing, New York.

“Affinity” refers to the strength of the sum total of noncovalentinteractions between a single binding site of a molecule (e.g., anantibody) and its binding partner (e.g., an antigen). Unless indicatedotherwise, as used herein, “binding affinity” refers to intrinsicbinding affinity which reflects a 1:1 interaction between members of abinding pair (e.g., antibody and antigen). The affinity of a molecule Xfor its partner Y can generally be represented by the dissociationconstant (Kd). Affinity can be measured by common methods known in theart, including those described herein. Specific illustrative andexemplary embodiments for measuring binding affinity are described inthe following.

The term “antibody” herein is used in the broadest sense andspecifically covers monoclonal antibodies, polyclonal antibodies,dimers, multimers, multispecific antibodies (e.g., bispecificantibodies), and antibody fragments, so long as they exhibit the desiredbiological activity (Miller et al. (2003) Jour. of Immunology170:4854-4861). Antibodies may be murine, human, humanized, chimeric, orderived from other species. An antibody is a protein generated by theimmune system that is capable of recognizing and binding to a specificantigen. (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) ImmunoBiology, 5th Ed., Garland Publishing, New York). A target antigengenerally has numerous binding sites, also called epitopes, recognizedby CDRs on multiple antibodies. Each antibody that specifically binds toa different epitope has a different structure. Thus, one antigen mayhave more than one corresponding antibody. An antibody includes afull-length immunoglobulin molecule or an immunologically active portionof a full-length immunoglobulin molecule, i.e., a molecule that containsan antigen binding site that immunospecifically binds an antigen of atarget of interest or part thereof, such targets including but notlimited to, cancer cell or cells that produce autoimmune antibodiesassociated with an autoimmune disease. The immunoglobulin disclosedherein can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class(e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass ofimmunoglobulin molecule. The immunoglobulins can be derived from anyspecies. In one aspect, however, the immunoglobulin is of human, murine,or rabbit origin.

“Antibody fragments” comprise a portion of a full length antibody,generally the antigen binding or variable region thereof. Examples ofantibody fragments include Fab, Fab′, F(ab′)2, and Fv fragments;diabodies; linear antibodies; minibodies (Olafsen et al. (2004) ProteinEng. Design & Sel. 17(4):315-323), fragments produced by a Fabexpression library, anti-idiotypic (anti-Id) antibodies, CDR(complementary determining region), and epitope-binding fragments of anydescribed herein which immunospecifically bind to cancer cell antigens,viral antigens or microbial antigens, single-chain antibody molecules;and multispecific antibodies formed from antibody fragments.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to polyclonalantibody preparations which include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody isdirected against a single determinant on the antigen. In addition totheir specificity, the monoclonal antibodies are advantageous in thatthey may be synthesized uncontaminated by other antibodies. The modifier“monoclonal” indicates the character of the antibody as being obtainedfrom a substantially homogeneous population of antibodies, and is not tobe construed as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by the hybridoma method firstdescribed by Kohler et al. (1975) Nature 256:495, or may be made byrecombinant DNA methods (see for example: U.S. Pat. No. 4,816,567; U.S.Pat. No. 5,807,715). The monoclonal antibodies may also be isolated fromphage antibody libraries using the techniques described in Clackson etal. (1991) Nature, 352:624-628; Marks et al. (1991) J. Mol. Biol.,222:581-597; for example.

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (U.S. Pat. No. 4,816,567; and Morrison etal. (1984) Proc. Natl. Acad. Sci. USA, 81:6851-6855). Chimericantibodies of interest herein include “primatized” antibodies comprisingvariable domain antigen-binding sequences derived from a non-humanprimate (e.g., Old World Monkey, Ape, etc.) and human constant regionsequences.

An “intact antibody” herein is one comprising a VL and VH domains, aswell as a light chain constant domain (CL) and heavy chain constantdomains, CH1, CH2 and CH3. The constant domains may be native sequenceconstant domains (e.g., human native sequence constant domains) or aminoacid sequence variant thereof. The intact antibody may have one or more“effector functions” which refer to those biological activitiesattributable to the Fc constant region (a native sequence Fc region oramino acid sequence variant Fc region) of an antibody. Examples ofantibody effector functions include C1q binding; complement dependentcytotoxicity; Fc receptor binding; antibody-dependent cell-mediatedcytotoxicity (ADCC); phagocytosis; and down regulation of cell surfacereceptors such as B cell receptor and BCR.

Depending on the amino acid sequence of the constant domain of theirheavy chains, intact antibodies can be assigned to different “classes.”There are five major classes of intact immunoglobulin antibodies: IgA,IgD, IgE, IgG, and IgM, and several of these may be further divided into“subclasses” (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.The heavy-chain constant domains that correspond to the differentclasses of antibodies are called α, δ, ε, γ, and μ, respectively. Thesubunit structures and three-dimensional configurations of differentclasses of immunoglobulins are well known. Ig forms includehinge-modifications or hingeless forms (Roux et al. (1998) J. Immunol.161:4083-4090; Lund et al. (2000) Eur. J. Biochem. 267:7246-7256; US2005/0048572; US 2004/0229310).

A “cysteine engineered antibody” or “cysteine engineered antibodyvariant” is an antibody in which one or more residues of an antibody aresubstituted with cysteine residues. In accordance with the presentdisclosure, the thiol group(s) of the cysteine engineered antibodies canbe conjugated to calicheamicin to form a THIOMAB™ antibody (i.e., aTHIOMAB™ drug conjugate (TDC), wherein in accordance with the presentdisclosure the drug is a calicheamicin derivative). In particularembodiments, the substituted residues occur at accessible sites of theantibody. By substituting those residues with cysteine, reactive thiolgroups are thereby positioned at accessible sites of the antibody andmay be used to conjugate the antibody to the drug moiety to create animmunoconjugate, as described further herein. For example, a THIOMAB™antibody may be an antibody with a single mutation of a non-cysteinenative residue to a cysteine in the light chain (e.g., G64C, K149C orR142C according to Kabat numbering) or in the heavy chain (e.g., D101Cor V184C or T205C according to Kabat numbering). In specific examples, aTHIOMAB™ antibody has a single cysteine mutation in either the heavy orlight chain such that each full-length antibody (i.e., an antibody withtwo heavy chains and two light chains) has two engineered cysteineresidues. Cysteine engineered antibodies and preparatory methods aredisclosed by US 2012/0121615 A1 (incorporated by reference herein in itsentirety).

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth/proliferation. Examples of cancer include, butare not limited to, carcinoma, lymphoma (e.g., Hodgkin's andnon-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. Moreparticular examples of such cancers include acute myeloid leukemia(AML), myelodysplastic syndrome (MDS), chronic myelogenous leukemia(CML), chronic myelomonocytic leukemia, acute promyelocytic leukemia(APL), chronic myeloproliferative disorder, thrombocytic leukemia,precursor B-cell acute lymphoblastic leukemia (pre-B-ALL), precursorT-cell acute lymphoblastic leukemia (preT-ALL), multiple myeloma (MM),mast cell disease, mast cell leukemia, mast cell sarcoma, myeloidsarcomas, lymphoid leukemia, and undifferentiated leukemia. In someembodiments, the cancer is myeloid leukemia. In some embodiments, thecancer is acute myeloid leukemia (AML).

The term “chimeric” antibody refers to an antibody in which a portion ofthe heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species.

The “class” of an antibody refers to the type of constant domain orconstant region possessed by its heavy chain. There are five majorclasses of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of thesemay be further divided into subclasses (isotypes), e.g., IgG₁, IgG₂,IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constant domains thatcorrespond to the different classes of immunoglobulins are called α, δ,ε, γ, and μ, respectively.

“Effector functions” refer to those biological activities attributableto the Fc region of an antibody, which vary with the antibody isotype.Examples of antibody effector functions include: C1q binding andcomplement dependent cytotoxicity (CDC); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g. B cell receptor); and B cellactivation.

An “effective amount” of an agent, e.g., a pharmaceutical formulation,refers to an amount effective, at dosages and for periods of timenecessary, to achieve the desired therapeutic or prophylactic result.

The term “epitope” refers to the particular site on an antigen moleculeto which an antibody binds. In some embodiments, the particular site onan antigen molecule to which an antibody binds is determined by hydroxylradical footprinting.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain that contains at least a portion of theconstant region. The term includes native sequence Fc regions andvariant Fc regions. In one embodiment, a human IgG heavy chain Fc regionextends from Cys226, or from Pro230, to the carboxyl-terminus of theheavy chain. However, the C-terminal lysine (Lys447) of the Fc regionmay or may not be present. Unless otherwise specified herein, numberingof amino acid residues in the Fc region or constant region is accordingto the EU numbering system, also called the EU index, as described inKabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.,1991.

“Framework” or “FR” refers to variable domain residues other thanhypervariable region (HVR) residues. The FR of a variable domaingenerally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in the followingsequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The terms “full length antibody,” “intact antibody,” and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a native antibody structure orhaving heavy chains that contain an Fc region as defined herein.

The terms “host cell,” “host cell line,” and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human or a human cellor derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues.

A “human consensus framework” is a framework which represents the mostcommonly occurring amino acid residues in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat et al., Sequences of Proteins of Immunological Interest, FifthEdition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In oneembodiment, for the VL, the subgroup is subgroup kappa I as in Kabat etal., supra. In one embodiment, for the VH, the subgroup is subgroup IIIas in Kabat et al., supra.

A “humanized” antibody refers to a chimeric antibody comprising aminoacid residues from non-human HVRs and amino acid residues from humanFRs. In certain embodiments, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the HVRs (e.g., CDRs) correspond tothose of a non-human antibody, and all or substantially all of the FRscorrespond to those of a human antibody. A humanized antibody optionallymay comprise at least a portion of an antibody constant region derivedfrom a human antibody. A “humanized form” of an antibody, e.g., anon-human antibody, refers to an antibody that has undergonehumanization.

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (VH and VL, respectively) of a native antibody generally havesimilar structures, with each domain comprising four conserved frameworkregions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindtet al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007).)A single VH or VL domain may be sufficient to confer antigen-bindingspecificity. Furthermore, antibodies that bind a particular antigen maybe isolated using a VH or VL domain from an antibody that binds theantigen to screen a library of complementary VL or VH domains,respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887(1993); Clarkson et al., Nature 352:624-628 (1991).

The term “vector,” as used herein, refers to a nucleic acid moleculecapable of propagating another nucleic acid to which it is linked. Theterm includes the vector as a self-replicating nucleic acid structure aswell as the vector incorporated into the genome of a host cell intowhich it has been introduced. Certain vectors are capable of directingthe expression of nucleic acids to which they are operatively linked.Such vectors are referred to herein as “expression vectors.”

The term “hypervariable region” or “HVR,” as used herein, refers to eachof the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops (“hypervariable loops”).Generally, native four-chain antibodies comprise six HVRs; three in theVH (H1, H2, H3), and three in the VL (L1, L2, L3). HVRs generallycomprise amino acid residues from the hypervariable loops and/or fromthe “complementarity determining regions” (CDRs), the latter being ofhighest sequence variability and/or involved in antigen recognition.Exemplary hypervariable loops occur at amino acid residues 26-32 (L1),50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3).(Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987).) Exemplary CDRs(CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) occur at amino acidresidues 24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 ofH2, and 95-102 of H3. (Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991).) With the exception of CDR1in VH, CDRs generally comprise the amino acid residues that form thehypervariable loops. CDRs also comprise “specificity determiningresidues,” or “SDRs,” which are residues that contact antigen. SDRs arecontained within regions of the CDRs called abbreviated-CDRs, or a-CDRs.Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, anda-CDR-H3) occur at amino acid residues 31-34 of L1, 50-55 of L2, 89-96of L3, 31-35B of H1, 50-58 of H2, and 95-102 of H3. (See Almagro andFransson, Front. Biosci. 13:1619-1633 (2008).) Unless otherwiseindicated, HVR residues and other residues in the variable domain (e.g.,FR residues) are numbered herein according to Kabat et al., supra.

An “individual” or “subject” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g., cows, sheep, cats, dogs, andhorses), primates (e.g., humans and non-human primates such as monkeys),rabbits, and rodents (e.g., mice and rats). In certain embodiments, theindividual or subject is a human.

An “isolated antibody” is one which has been separated from a componentof its natural environment. In some embodiments, an antibody is purifiedto greater than 95% or 99% purity as determined by, for example,electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillaryelectrophoresis) or chromatographic (e.g., ion exchange or reverse phaseHPLC). For review of methods for assessment of antibody purity, see,e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

“Native antibodies” refer to naturally occurring immunoglobulinmolecules with varying structures. For example, native IgG antibodiesare heterotetrameric glycoproteins of about 150,000 daltons, composed oftwo identical light chains and two identical heavy chains that aredisulfide-bonded. From N- to C-terminus, each heavy chain has a variableregion (VH), also called a variable heavy domain or a heavy chainvariable domain, followed by three constant domains (CH1, CH2, and CH3).Similarly, from N- to C-terminus, each light chain has a variable region(VL), also called a variable light domain or a light chain variabledomain, followed by a constant light (CL) domain. The light chain of anantibody may be assigned to one of two types, called kappa (κ) andlambda (λ), based on the amino acid sequence of its constant domain.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, including digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows: 100 times thefraction X/Y where X is the number of amino acid residues scored asidentical matches by the sequence alignment program ALIGN-2 in thatprogram's alignment of A and B, and where Y is the total number of aminoacid residues in B. It will be appreciated that where the length ofamino acid sequence A is not equal to the length of amino acid sequenceB, the % amino acid sequence identity of A to B will not equal the %amino acid sequence identity of B to A. Unless specifically statedotherwise, all % amino acid sequence identity values used herein areobtained as described in the immediately preceding paragraph using theALIGN-2 computer program.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical formulation, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

The terms “treat” and “treatment” refer to both therapeutic treatmentand prophylactic or preventative measures, wherein the object is toprevent or slow down (lessen) an undesired physiological change ordisorder, such as the development or spread of cancer. For purposes ofthis invention, beneficial or desired clinical results include, but arenot limited to, alleviation of symptoms, diminishment of extent ofdisease, stabilized (i.e., not worsening) state of disease, delay orslowing of disease progression, amelioration or palliation of thedisease state, and remission (whether partial or total), whetherdetectable or undetectable. “Treatment” can also mean prolongingsurvival as compared to expected survival if not receiving treatment.Those in need of treatment include those already with the condition ordisorder as well as those prone to have the condition or disorder orthose in which the condition or disorder is to be prevented.

The term “therapeutically effective amount” refers to an amount of adrug effective to treat a disease or disorder in a mammal. In the caseof cancer, the therapeutically effective amount of the drug may reducethe number of cancer cells; reduce the tumor size; inhibit (i.e., slowto some extent and preferably stop) cancer cell infiltration intoperipheral organs; inhibit (i.e., slow to some extent and preferablystop) tumor metastasis; inhibit, to some extent, tumor growth; and/orrelieve to some extent one or more of the symptoms associated with thecancer. To the extent the drug may prevent growth and/or kill existingcancer cells, it may be cytostatic and/or cytotoxic. For cancer therapy,efficacy can, for example, be measured by assessing the time to diseaseprogression (TTP) and/or determining the response rate (RR).

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. A “tumor” comprises one or more cancerouscells. Examples of cancer include, but are not limited to, carcinoma,lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. Moreparticular examples of such cancers include squamous cell cancer (e.g.,epithelial squamous cell cancer), lung cancer including small-cell lungcancer, non-small cell lung cancer (“NSCLC”), adenocarcinoma of the lungand squamous carcinoma of the lung, cancer of the peritoneum,hepatocellular cancer, gastric or stomach cancer includinggastrointestinal cancer, pancreatic cancer, glioblastoma, cervicalcancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breastcancer, colon cancer, rectal cancer, colorectal cancer, endometrial oruterine carcinoma, salivary gland carcinoma, kidney or renal cancer,prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, analcarcinoma, penile carcinoma, as well as head and neck cancer.

The term “leaving group,” as used herein, refers to a sulfhydryl moietythat leaves in the course of a chemical reaction involving the groups asdescribed herein.

The term “hydrocarbyl” as used herein describes organic compounds orradicals consisting exclusively of the elements carbon and hydrogen.These moieties include, without limitation, alkyl, alkenyl, alkynyl, andaryl moieties. These moieties also include alkyl, alkenyl, alkynyl, andaryl moieties substituted with other aliphatic or cyclic hydrocarbongroups, such as alkaryl, alkenaryl and alkynaryl. Unless otherwiseindicated, these moieties preferably comprise 1 to 20 carbon atoms, 1 to10 carbon atoms or 1 to 6 carbon atoms.

Unless otherwise indicated, the alkyl groups described herein arepreferably lower alkyl containing from one to eight carbon atoms in theprincipal chain. They may be straight or branched chain or cyclicincluding, but not limited to, methyl, ethyl, propyl, isopropyl, allyl,benzyl, hexyl and the like.

Unless otherwise indicated, the alkynyl groups described herein arepreferably lower alkynyl containing from two to eight carbon atoms inthe principal chain and up to 20 carbon atoms. They may be straight orbranched chain including, but not limited to, ethynyl, propynyl,butynyl, isobutynyl, hexynyl, and the like.

The term “aryl” as used herein alone or as part of another group denotesoptionally substituted homocyclic aromatic groups, preferably monocyclicor bicyclic groups containing from 5 to 20 carbons, from 5 to 10carbons, or from 5 to 6 carbons in the ring portion, including, but notlimited to, phenyl, biphenyl, naphthyl, substituted phenyl, substitutedbiphenyl or substituted naphthyl. The aryl moieties may optionallycomprise one or more hetero atoms selected from O, S and N. Suchheteroaromatics may comprise 1 or 2 nitrogen atoms, 1 or 2 sulfur atoms,1 or 2 oxygen atoms, and combinations thereof, in the ring, wherein theeach hetero atom is bonded to the remainder of the molecule through acarbon. Non limiting exemplary groups include pyridine, pyrazine,pyrimidine, pyrazole, pyrrole, imidazole, thiopene, thiopyrrilium,parathiazine, indole, purine, benzimidazole, quinolone, phenothiazine.Non-limiting exemplary substituents include one or more of the followinggroups: hydrocarbyl, substituted hydrocarbyl, alkyl, alkoxy, acyl,acyloxy, alkenyl, alkenoxy, aryl, aryloxy, amino, amido, acetal,carbamyl, carbocyclo, cyano, ester, ether, halogen, heterocyclo,hydroxy, keto, ketal, phospho, nitro, and thio.

The “substituted” moieties described herein are moieties such ashydrocarbyl, alkyl and aryl which are substituted with at least one atomother than carbon, including moieties in which a carbon chain atom issubstituted with a hetero atom such as nitrogen, oxygen, silicon,phosphorous, boron, sulfur, or a halogen atom. These substituentsinclude, but are not limited to, halogen, heterocyclo, alkoxy, alkenoxy,alkynoxy, aryloxy, hydroxy, keto, acyl, acyloxy, nitro, tertiary amino,amido, nitro, cyano, thio, sulfinate, sulfonamide, ketals, acetals,esters and ethers.

The terms “halogen” as used herein alone or as part of another grouprefer to chlorine, bromine, fluorine, and iodine.

Antibodies

In any of the embodiments of the disclosure, an antibody is humanized.In one embodiment, an antibody comprises HVRs as in any of theembodiments of the disclosure, and further comprises a human acceptorframework, e.g. a human immunoglobulin framework or a human consensusframework. In certain embodiments, the human acceptor framework is thehuman VL kappa I consensus (VLKI) framework and/or the VH framework VH1.In certain embodiments, the human acceptor framework is the human VLkappa I consensus (VLKI) framework and/or the VH framework VH1comprising any one of the following mutations.

In another aspect, the antibody comprises a VH as in any of theembodiments provided herein, and a VL as in any of the embodimentsprovided herein.

In a further aspect of the invention, an antibody according to any ofthe embodiments herein is a monoclonal antibody, including a humanantibody. In one embodiment, an antibody is an antibody fragment, e.g.,a Fv, Fab, Fab′, scFv, diabody, or F(ab′)2 fragment. In anotherembodiment, the antibody is a substantially full length antibody, e.g.,an IgG1 antibody, IgG2a antibody or other antibody class or isotype asdefined herein.

In a further aspect, an antibody according to any of the embodimentsherein may incorporate any of the features, singly or in combination, asdescribed herein.

1. Antibody Affinity

In certain embodiments, an antibody provided herein has a dissociationconstant (Kd) of ≦1 μM, ≦100 nM, ≦50 nM, ≦10 nM, ≦5 nM, ≦1 nM, ≦0.1 nM,≦0.01 nM, or ≦0.001 nM, and optionally is ≧10⁻¹³ M. (e.g. 10⁻⁸M or less,e.g. from 10⁻⁸M to 10⁻¹³ M, e.g., from 10⁻⁹M to 10⁻¹³ M).

In one embodiment, Kd is measured by a radiolabeled antigen bindingassay (RIA) performed with the Fab version of an antibody of interestand its antigen as described by the following assay. Solution bindingaffinity of Fabs for antigen is measured by equilibrating Fab with aminimal concentration of (¹²⁵I)-labeled antigen in the presence of atitration series of unlabeled antigen, then capturing bound antigen withan anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol.293:865-881(1999)). To establish conditions for the assay, MICROTITER®multi-well plates (Thermo Scientific) are coated overnight with 5 μg/mlof a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate(pH 9.6), and subsequently blocked with 2% (w/v) bovine serum albumin inPBS for two to five hours at room temperature (approximately 23° C.). Ina non-adsorbent plate (Nunc #269620), 100 pM or 26 pM [¹²⁵I]-antigen aremixed with serial dilutions of a Fab of interest (e.g., consistent withassessment of the anti-VEGF antibody, Fab-12, in Presta et al., CancerRes. 57:4593-4599 (1997)). The Fab of interest is then incubatedovernight; however, the incubation may continue for a longer period(e.g., about 65 hours) to ensure that equilibrium is reached.Thereafter, the mixtures are transferred to the capture plate forincubation at room temperature (e.g., for one hour). The solution isthen removed and the plate washed eight times with 0.1% polysorbate 20(TWEEN-20®) in PBS. When the plates have dried, 150 μl/well ofscintillant (MICROSCINT-20™; Packard) is added, and the plates arecounted on a TOPCOUNT™ gamma counter (Packard) for ten minutes.Concentrations of each Fab that give less than or equal to 20% ofmaximal binding are chosen for use in competitive binding assays.

According to another embodiment, Kd is measured using surface plasmonresonance assays using a BIACORE®-2000, BAICORE®-T200 or a BIACORE®-3000(BIAcore, Inc., Piscataway, N.J.) at 25° C. with immobilized antigen CMSchips at ˜10 response units (RU). Briefly, carboxymethylated dextranbiosensor chips (CMS, BIACORE, Inc.) are activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 μg/ml (˜0.2μM) and/or HBS-P (0.01 M Hepes pH7.4, 0.15M NaCl, 0.005% Surfactant P20)before injection at a flow rate of 5 μl/minute and/or 30 μl/minute toachieve approximately 10 response units (RU) of coupled protein.Following the injection of antigen, 1 M ethanolamine is injected toblock unreacted groups. For kinetics measurements, two-fold serialdilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05%polysorbate 20 (TWEEN-20™2) surfactant (PBST) at 25° C. at a flow rateof approximately 25 μl/min. Association rates (k_(on)) and dissociationrates (k_(off)) are calculated using a simple one-to-one Langmuirbinding model (BIACORE® Evaluation Software version 3.2) bysimultaneously fitting the association and dissociation sensorgrams. Theequilibrium dissociation constant (Kd) is calculated as the ratiok_(off)/k_(on). See, e.g., Chen et al., J. Mol. Biol. 293:865-881(1999). If the on-rate exceeds 10⁶ M⁻¹ s⁻¹ by the surface plasmonresonance assay describe herein, then the on-rate can be determined byusing a fluorescent quenching technique that measures the increase ordecrease in fluorescence emission intensity (excitation=295 nm;emission=340 nm, 16 nm band-pass) at 25° C. of a 20 nM anti-antigenantibody (Fab form) in PBS, pH 7.2, in the presence of increasingconcentrations of antigen as measured in a spectrometer, such as astop-flow equipped spectrophometer (Aviv Instruments) or a 8000-seriesSLM-AMINCO spectrophotometer (ThermoSpectronic) with a stirred cuvette.

2. Antibody Fragments

In certain embodiments, an antibody provided herein is an antibodyfragment. Antibody fragments include, but are not limited to, Fab, Fab′,Fab′-SH, F(ab′)2, Fv, and scFv fragments, and other fragments describedherein. For a review of certain antibody fragments, see Hudson et al.Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g.,Pluckthün, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315(1994); see also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and5,587,458. For discussion of Fab and F(ab′)2 fragments comprisingsalvage receptor binding epitope residues and having increased in vivohalf-life, see U.S. Pat. No. 5,869,046.

Diabodies are antibody fragments with two antigen-binding sites that maybe bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161;Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc.Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodiesare also described in Hudson et al., Nat. Med. 9:129-134 (2003).

Single-domain antibodies are antibody fragments comprising all or aportion of the heavy chain variable domain or all or a portion of thelight chain variable domain of an antibody. In certain embodiments, asingle-domain antibody is a human single-domain antibody (Domantis,Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B1).

Antibody fragments can be made by various techniques, including but notlimited to proteolytic digestion of an intact antibody as well asproduction by recombinant host cells (e.g. E. coli or phage), asdescribed herein.

3. Chimeric and Humanized Antibodies

In certain embodiments, an antibody provided herein is a chimericantibody. Certain chimeric antibodies are described, e.g., in U.S. Pat.No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA,81:6851-6855 (1984)). In one example, a chimeric antibody comprises anon-human variable region (e.g., a variable region derived from a mouse,rat, hamster, rabbit, or non-human primate, such as a monkey) and ahuman constant region. In a further example, a chimeric antibody is a“class switched” antibody in which the class or subclass has beenchanged from that of the parent antibody. Chimeric antibodies includeantigen-binding fragments thereof.

In certain embodiments, a chimeric antibody is a humanized antibody.Typically, a non-human antibody is humanized to reduce immunogenicity tohumans, while retaining the specificity and affinity of the parentalnon-human antibody. Generally, a humanized antibody comprises one ormore variable domains in which HVRs, e.g., CDRs, (or portions thereof)are derived from a non-human antibody, and FRs (or portions thereof) arederived from human antibody sequences. A humanized antibody optionallywill also comprise at least a portion of a human constant region. Insome embodiments, some FR residues in a humanized antibody aresubstituted with corresponding residues from a non-human antibody (e.g.,the antibody from which the HVR residues are derived), e.g., to restoreor improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, e.g., inAlmagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and arefurther described, e.g., in Riechmann et al., Nature 332:323-329 (1988);Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S.Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri etal., Methods 36:25-34 (2005) (describing SDR (a-CDR) grafting); Padlan,Mol. Immunol. 28:489-498 (1991) (describing “resurfacing”); Dall'Acquaet al., Methods 36:43-60 (2005) (describing “FR shuffling”); and Osbournet al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer,83:252-260 (2000) (describing the “guided selection” approach to FRshuffling).

Human framework regions that may be used for humanization include butare not limited to: framework regions selected using the “best-fit”method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); frameworkregions derived from the consensus sequence of human antibodies of aparticular subgroup of light or heavy chain variable regions (see, e.g.,Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta etal. J. Immunol., 151:2623 (1993)); human mature (somatically mutated)framework regions or human germline framework regions (see, e.g.,Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and frameworkregions derived from screening FR libraries (see, e.g., Baca et al., J.Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem.271:22611-22618 (1996)).

4. Human Antibodies

In certain embodiments, an antibody provided herein is a human antibody.Human antibodies can be produced using various techniques known in theart. Human antibodies are described generally in van Dijk and van deWinkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin.Immunol. 20:450-459 (2008).

Human antibodies may be prepared by administering an immunogen to atransgenic animal that has been modified to produce intact humanantibodies or intact antibodies with human variable regions in responseto antigenic challenge. Such animals typically contain all or a portionof the human immunoglobulin loci, which replace the endogenousimmunoglobulin loci, or which are present extrachromosomally orintegrated randomly into the animal's chromosomes. In such transgenicmice, the endogenous immunoglobulin loci have generally beeninactivated. For review of methods for obtaining human antibodies fromtransgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). Seealso, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 describing XENOMOUSE™technology; U.S. Pat. No. 5,770,429 describing HUMAB® technology; U.S.Pat. No. 7,041,870 describing K-M MOUSE® technology, and U.S. PatentApplication Publication No. US 2007/0061900, describing VELoCIMOUSE®technology). Human variable regions from intact antibodies generated bysuch animals may be further modified, e.g., by combining with adifferent human constant region.

Human antibodies can also be made by hybridoma-based methods. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described. (See, e.g., Kozbor J.Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987); and Boemer et al., J. Immunol., 147: 86 (1991).) Humanantibodies generated via human B-cell hybridoma technology are alsodescribed in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562(2006). Additional methods include those described, for example, in U.S.Pat. No. 7,189,826 (describing production of monoclonal human IgMantibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue,26(4):265-268 (2006) (describing human-human hybridomas). Humanhybridoma technology (Trioma technology) is also described in Vollmersand Brandlein, Histology and Histopathology, 20(3):927-937 (2005) andVollmers and Brandlein, Methods and Findings in Experimental andClinical Pharmacology, 27(3): 185-91 (2005).

Human antibodies may also be generated by isolating Fv clone variabledomain sequences selected from human-derived phage display libraries.Such variable domain sequences may then be combined with a desired humanconstant domain. Techniques for selecting human antibodies from antibodylibraries are described herein.

5. Library-Derived Antibodies

Antibodies of the invention may be isolated by screening combinatoriallibraries for antibodies with the desired activity or activities. Forexample, a variety of methods are known in the art for generating phagedisplay libraries and screening such libraries for antibodies possessingthe desired binding characteristics. Such methods are reviewed, e.g., inHoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien etal., ed., Human Press, Totowa, N.J., 2001) and further described, e.g.,in the McCafferty et al., Nature 348:552-554; Clackson et al., Nature352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992);Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo,ed., Human Press, Totowa, N.J., 2003); Sidhu et al., J Mol. Biol.338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093(2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472(2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132(2004).

In certain phage display methods, repertoires of VH and VL genes areseparately cloned by polymerase chain reaction (PCR) and recombinedrandomly in phage libraries, which can then be screened forantigen-binding phage as described in Winter et al., Ann. Rev. Immunol.,12: 433-455 (1994). Phage typically display antibody fragments, eitheras single-chain Fv (scFv) fragments or as Fab fragments. Libraries fromimmunized sources provide high-affinity antibodies to the immunogenwithout the requirement of constructing hybridomas. Alternatively, thenaive repertoire can be cloned (e.g., from human) to provide a singlesource of antibodies to a wide range of non-self and also self antigenswithout any immunization as described by Griffiths et al., EMBO J, 12:725-734 (1993). Finally, naive libraries can also be made syntheticallyby cloning unrearranged V-gene segments from stem cells, and using PCRprimers containing random sequence to encode the highly variable CDR3regions and to accomplish rearrangement in vitro, as described byHoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patentpublications describing human antibody phage libraries include, forexample: U.S. Pat. No. 5,750,373, and US Patent Publication Nos.2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598,2007/0237764, 2007/0292936, and 2009/0002360.

Antibodies or antibody fragments isolated from human antibody librariesare considered human antibodies or human antibody fragments herein.

6. Multispecific Antibodies

In certain embodiments, an antibody provided herein is a multispecificantibody, e.g. a bispecific antibody. The term “multispecific antibody”is used in the broadest sense and specifically covers an antibodycomprising an antigen-binding domain that has polyepitopic specificity(i.e., is capable of specifically binding to two, or more, differentepitopes on one biological molecule or is capable of specificallybinding to epitopes on two, or more, different biological molecules). Insome embodiments, multispecific antibodies are monoclonal antibodiesthat have binding specificities for at least two different sites. Insome embodiments, an antigen-binding domain of a multispecific antibody(such as a bispecific antibody) comprises two VH/VL units, wherein afirst VH/VL unit specifically binds to a first epitope and a secondVH/VL unit specifically binds to a second epitope, wherein each VH/VLunit comprises a heavy chain variable domain (VH) and a light chainvariable domain (VL). Such multispecific antibodies include, but are notlimited to, full length antibodies, antibodies having two or more VL andVH domains, antibody fragments such as Fab, Fv, dsFv, scFv, diabodies,bispecific diabodies and triabodies, antibody fragments that have beenlinked covalently or non-covalently. A VH/VL unit that further comprisesat least a portion of a heavy chain variable region and/or at least aportion of a light chain variable region may also be referred to as an“arm” or “hemimer” or “half antibody.” In some embodiments, a hemimercomprises a sufficient portion of a heavy chain variable region to allowintramolecular disulfide bonds to be formed with a second hemimer. Insome embodiments, a hemimer comprises a knob mutation or a holemutation, for example, to allow heterodimerization with a second hemimeror half antibody that comprises a complementary hole mutation or knobmutation. Knob mutations and hole mutations are discussed furtherherein.

In certain embodiments, a multispecific antibody provided herein may bea bispecific antibody. The term “bispecific antibody” is used in thebroadest sense and covers a multispecific antibody comprising anantigen-binding domain that is capable of specifically binding to twodifferent epitopes on one biological molecule or is capable ofspecifically binding to epitopes on two different biological molecules.A bispecific antibody may also be referred to herein as having “dualspecificity” or as being “dual specific.” Bispecific antibodies can beprepared as full length antibodies or antibody fragments. The term“biparatopic antibody” as used herein, refers to a bispecific antibodywhere a first antigen-binding domain and a second antigen-binding domainbind to two different epitopes on the same antigen molecule or it maybind to epitopes on two different antigen molecules.

In some embodiments, the first antigen-binding domain and the secondantigen-binding domain of the biparatopic antibody may bind the twoepitopes within one and the same antigen molecule (intramolecularbinding). For example, the first antigen-binding domain and the secondantigen-binding domain of the biparatopic antibody may bind to twodifferent epitopes on the same antibody molecule. In certainembodiments, the two different epitopes that a biparatopic antibodybinds are epitopes that are not normally bound at the same time by onemonospecific antibody, such as e.g. a conventional antibody or oneimmunoglobulin single variable domain.

In some embodiments, the first antigen-binding domain and the secondantigen-binding domain of the biparatopic antibody may bind epitopeslocated within two distinct antigen molecules.

Techniques for making multispecific antibodies include, but are notlimited to, recombinant co-expression of two immunoglobulin heavychain-light chain pairs having different specificities (see Milstein andCuello, Nature 305: 537 (1983)), WO 93/08829, and Traunecker et al.,EMBO J. 10: 3655 (1991)), and “knob-in-hole” engineering (see, e.g.,U.S. Pat. No. 5,731,168), WO2009/089004, US2009/0182127, US2011/0287009,Marvin and Zhu, Acta Pharmacol. Sin. (2005) 26(6):649-658, andKontermann (2005) Acta Pharmacol. Sin., 26:1-9). The term“knob-into-hole” or “KnH” technology as used herein refers to thetechnology directing the pairing of two polypeptides together in vitroor in vivo by introducing a protuberance (knob) into one polypeptide anda cavity (hole) into the other polypeptide at an interface in which theyinteract. For example, KnHs have been introduced in the Fc:Fc bindinginterfaces, CL:CH1 interfaces or VH/VL interfaces of antibodies (see,e.g., US 2011/0287009, US2007/0178552, WO 96/027011, WO 98/050431, andZhu et al., 1997, Protein Science 6:781-788). In some embodiments, KnHsdrive the pairing of two different heavy chains together during themanufacture of multispecific antibodies. For example, multispecificantibodies having KnH in their Fc regions can further comprise singlevariable domains linked to each Fc region, or further comprise differentheavy chain variable domains that pair with similar or different lightchain variable domains. KnH technology can be also be used to pair twodifferent receptor extracellular domains together or any otherpolypeptide sequences that comprises different target recognitionsequences (e.g., including affibodies, peptibodies and other Fcfusions).

The term “knob mutation” as used herein refers to a mutation thatintroduces a protuberance (knob) into a polypeptide at an interface inwhich the polypeptide interacts with another polypeptide. In someembodiments, the other polypeptide has a hole mutation.

A “protuberance” refers to at least one amino acid side chain whichprojects from the interface of a first polypeptide and is thereforepositionable in a compensatory cavity in the adjacent interface (i.e.the interface of a second polypeptide) so as to stabilize theheteromultimer, and thereby favor heteromultimer formation overhomomultimer formation, for example. The protuberance may exist in theoriginal interface or may be introduced synthetically (e.g. by alteringnucleic acid encoding the interface). In some embodiments, nucleic acidencoding the interface of the first polypeptide is altered to encode theprotuberance. To achieve this, the nucleic acid encoding at least one“original” amino acid residue in the interface of the first polypeptideis replaced with nucleic acid encoding at least one “import” amino acidresidue which has a larger side chain volume than the original aminoacid residue. It will be appreciated that there can be more than oneoriginal and corresponding import residue. The side chain volumes of thevarious amino residues are shown, for example, in Table 1 ofUS2011/0287009. A mutation to introduce a “protuberance” may be referredto as a “knob mutation.”

In some embodiments, import residues for the formation of a protuberanceare naturally occurring amino acid residues selected from arginine (R),phenylalanine (F), tyrosine (Y) and tryptophan (W). In some embodiments,an import residue is tryptophan or tyrosine. In some embodiment, theoriginal residue for the formation of the protuberance has a small sidechain volume, such as alanine, asparagine, aspartic acid, glycine,serine, threonine or valine.

A “cavity” refers to at least one amino acid side chain which isrecessed from the interface of a second polypeptide and thereforeaccommodates a corresponding protuberance on the adjacent interface of afirst polypeptide. The cavity may exist in the original interface or maybe introduced synthetically (e.g. by altering nucleic acid encoding theinterface). In some embodiments, nucleic acid encoding the interface ofthe second polypeptide is altered to encode the cavity. To achieve this,the nucleic acid encoding at least one “original” amino acid residue inthe interface of the second polypeptide is replaced with DNA encoding atleast one “import” amino acid residue which has a smaller side chainvolume than the original amino acid residue. It will be appreciated thatthere can be more than one original and corresponding import residue. Insome embodiments, import residues for the formation of a cavity arenaturally occurring amino acid residues selected from alanine (A),serine (S), threonine (T) and valine (V). In some embodiments, an importresidue is serine, alanine or threonine. In some embodiments, theoriginal residue for the formation of the cavity has a large side chainvolume, such as tyrosine, arginine, phenylalanine or tryptophan. Amutation to introduce a “cavity” may be referred to as a “holemutation.”

The protuberance is “positionable” in the cavity which means that thespatial location of the protuberance and cavity on the interface of afirst polypeptide and second polypeptide respectively and the sizes ofthe protuberance and cavity are such that the protuberance can belocated in the cavity without significantly perturbing the normalassociation of the first and second polypeptides at the interface. Sinceprotuberances such as Tyr, Phe and Trp do not typically extendperpendicularly from the axis of the interface and have preferredconformations, the alignment of a protuberance with a correspondingcavity may, in some instances, rely on modeling the protuberance/cavitypair based upon a three-dimensional structure such as that obtained byX-ray crystallography or nuclear magnetic resonance (NMR). This can beachieved using widely accepted techniques in the art.

In some embodiments, a knob mutation in an IgG1 constant region isT366W. In some embodiments, a hole mutation in an IgG1 constant regioncomprises one or more mutations selected from T366S, L368A and Y407V. Insome embodiments, a hole mutation in an IgG1 constant region comprisesT366S, L368A and Y407V.

In some embodiments, a knob mutation in an IgG4 constant region isT366W. In some embodiments, a hole mutation in an IgG4 constant regioncomprises one or more mutations selected from T366S, L368A, and Y407V.In some embodiments, a hole mutation in an IgG4 constant regioncomprises T366S, L368A, and Y407V.

Multi-specific antibodies may also be made by engineering electrostaticsteering effects for making antibody Fc-heterodimeric molecules (WO2009/089004A1); cross-linking two or more antibodies or fragments (see,e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science, 229: 81(1985)); using leucine zippers to produce bi-specific antibodies (see,e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); using“diabody” technology for making bispecific antibody fragments (Hollingeret al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and usingsingle-chain Fv (sFv) dimers (Gruber et al., J. Immunol., 152:5368(1994)); and preparing trispecific antibodies as described, e.g., inTutt et al. J. Immunol. 147: 60 (1991).

Engineered antibodies with three or more functional antigen bindingsites, including “Octopus antibodies” or “dual-variable domainimmunoglobulins” (DVDs) are also included herein (US 2006/0025576A1, andWu et al. (2007) Nature Biotechnology).

In certain embodiments, one or more amino acid modifications may beintroduced into the Fc region of an antibody provided herein, therebygenerating an Fc region variant. The Fc region variant may comprise ahuman Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fcregion) comprising an amino acid modification (e.g. a substitution) atone or more amino acid positions.

In certain embodiments, the invention contemplates an antibody variantthat possesses some but not all effector functions, which make it adesirable candidate for applications in which the half life of theantibody in vivo is important yet certain effector functions (such ascomplement and ADCC) are unnecessary or deleterious. In vitro and/or invivo cytotoxicity assays can be conducted to confirm thereduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theantibody lacks FcγR binding (hence likely lacking ADCC activity), butretains FcRn binding ability. The primary cells for mediating ADCC, NKcells, express Fc(RIII only, whereas monocytes express Fc(RI, Fc(RII andFc(RIII. FcR expression on hematopoietic cells is summarized in Table 3on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991).Non-limiting examples of in vitro assays to assess ADCC activity of amolecule of interest is described in U.S. Pat. No. 5,500,362 (see, e.g.Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) andHellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985);5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361(1987)). Alternatively, non-radioactive assays methods may be employed(see, for example, ACTI™ non-radioactive cytotoxicity assay for flowcytometry (CellTechnology, Inc. Mountain View, Calif.; and CytoTox 96®non-radioactive cytotoxicity assay (Promega, Madison, Wis.). Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and Natural Killer (NK) cells. Alternatively, oradditionally, ADCC activity of the molecule of interest may be assessedin vivo, e.g., in an animal model such as that disclosed in Clynes etal. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays mayalso be carried out to confirm that the antibody is unable to bind C1qand hence lacks CDC activity. See, e.g., C1q and C3c binding ELISA in WO2006/029879 and WO 2005/100402. To assess complement activation, a CDCassay may be performed (see, for example, Gazzano-Santoro et al., J.Immunol. Methods 202:163 (1996); Cragg, M. S. et al., Blood101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie, Blood103:2738-2743 (2004)). FcRn binding and in vivo clearance/half lifedeterminations can also be performed using methods known in the art(Petkova, S. B. et al., Intl. Immunol. 18(12):1759-1769 (2006)).

Antibodies with reduced effector function include those withsubstitution of one or more of Fc region residues 238, 265, 269, 270,297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fcmutants with substitutions at two or more of amino acid positions 265,269, 270, 297 and 327, including the so-called “DANA” Fc mutant withsubstitution of residues 265 and 297 to alanine (U.S. Pat. No.7,332,581).

In certain embodiments Pro329 of a wild-type human Fc region issubstituted with glycine or arginine or an amino acid residue largeenough to destroy the proline sandwich within the Fc/Fcγ receptorinterface, that is formed between the proline329 of the Fc andtryptophan residues Trp 87 and Trp 110 of FcgRIII (Sondermann et al.:Nature 406, 267-273 (20 Jul. 2000)). In a further embodiment, at leastone further amino acid substitution in the Fc variant is S228P, E233P,L234A, L235A, L235E, N297A, N297D, or P331S and still in anotherembodiment said at least one further amino acid substitution is L234Aand L235A of the human IgG1 Fc region or S228P and L235E of the humanIgG4 Fc region (U.S. Pat. No. 8,969,526 which is incorporated byreference in its entirety).

In certain embodiments, a polypeptide comprises the Fc variant of awild-type human IgG Fc region wherein the polypeptide has Pro329 of thehuman IgG Fc region substituted with glycine and wherein the Fc variantcomprises at least two further amino acid substitutions at L234A andL235A of the human IgG1 Fc region or S228P and L235E of the human IgG4Fc region, and wherein the residues are numbered according to the EUindex of Kabat (U.S. Pat. No. 8,969,526 which is incorporated byreference in its entirety). In certain embodiments, the polypeptidecomprising the P329G, L234A and L235A substitutions exhibit a reducedaffinity to the human FcγRIIIA and FcγRIIA, for down-modulation of ADCCto at least 20% of the ADCC induced by the polypeptide comprising thewild type human IgG Fc region, and/or for down-modulation of ADCP (U.S.Pat. No. 8,969,526 which is incorporated by reference in its entirety).

In a specific embodiment the polypeptide comprising an Fc variant of awild type human Fc polypeptide comprises a triple mutation: an aminoacid substitution at position Pro329, a L234A and a L235A mutation(P329/LALA) (U.S. Pat. No. 8,969,526 which is incorporated by referencein its entirety). In specific embodiments, the polypeptide comprises thefollowing amino acid substitutions: P329G, L234A, and L235A.

Certain antibody variants with improved or diminished binding to FcRsare described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, andShields et al., J. Biol. Chem. 9(2): 6591-6604 (2001)).

In certain embodiments, an antibody variant comprises an Fc region withone or more amino acid substitutions which improve ADCC, e.g.,substitutions at positions 298, 333, and/or 334 of the Fc region (EUnumbering of residues).

In some embodiments, alterations are made in the Fc region that resultin altered (i.e., either improved or diminished) C1q binding and/orComplement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat.No. 6,194,551, WO 99/51642, and Idusogie et al., J. Immunol. 164:4178-4184 (2000).

Antibodies with increased half-lives and improved binding to theneonatal Fc receptor (FcRn), which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) andKim et al., J. Immunol. 24:249 (1994)), are described inUS2005/0014934A1 (Hinton et al.). Those antibodies comprise an Fc regionwith one or more substitutions therein which improve binding of the Fcregion to FcRn. Such Fc variants include those with substitutions at oneor more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307,311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434,e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826).

See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Pat. No.5,648,260; U.S. Pat. No. 5,624,821; and WO 94/29351 concerning otherexamples of Fc region variants.

7. Cysteine Engineered Antibody Variants

In certain embodiments, it may be desirable to create cysteineengineered antibodies, e.g., “THIOMAB™ antibody,” in which one or moreresidues of an antibody are substituted with cysteine residues. Inparticular embodiments, the substituted residues occur at accessiblesites of the antibody. By substituting those residues with cysteine,reactive thiol groups are thereby positioned at accessible sites of theantibody and may be used to conjugate the antibody to the drug moiety tocreate an immunoconjugate, as described further herein. In certainembodiments, any one or more of the following residues may besubstituted with cysteine: V205 (Kabat numbering) of the light chain;K149 (Kabat numbering) of the light chain; A118 (EU numbering) of theheavy chain; and 5400 (EU numbering) of the heavy chain Fc region.Cysteine engineered antibodies may be generated as described, e.g., inU.S. Pat. No. 7,521,541.

In some aspects, a THIOMAB™ antibody comprises one of the heavy or lightchain cysteine substitutions listed in Table 1 below.

TABLE 1 Screening GNE Kabat Chain Mutation Mutation Mutation (HC/LC)Residue Site # Site # Site # LC T 22 22 22 LC K 39 39 39 LC Y 49 49 49LC Y 55 55 55 LC T 85 85 85 LC T 97 97 97 LC I 106 106 106 LC R 108 108108 LC R 142 142 142 LC K 149 149 149 LC V 205 205 205 HC T 117 114 110HC A 143 140 136 HC L 177 174 170 HC L 182 179 175 HC T 190 187 183 HC T212 209 205 HC V 265 262 258 HC G 374 371 367 HC Y 376 373 369 HC E 385382 378 HC S 427 424 420 HC N 437 434 430 HC Q 441 438 434

In other aspects, a THIOMAB™ antibody comprises one of the heavy chaincysteine substitutions listed in Table 2.

TABLE 2 Screening GNE Kabat Chain Mutation Mutation Mutation (HC/LC)Residue Site # Site # Site # HC T 117 114 110 HC A 143 140 136 HC L 177174 170 HC L 182 179 175 HC T 190 187 183 HC T 212 209 205 HC V 265 262258 HC G 374 371 367 HC Y 376 373 369 HC E 385 382 378 HC S 427 424 420HC N 437 434 430 HC Q 441 438 434

In some other aspects, a THIOMAB™ antibody comprises one of the lightchain cysteine substitutions listed in Table 3.

TABLE 3 Screening GNE Kabat Chain Mutation Mutation Mutation (HC/LC)Residue Site # Site # Site # LC I 106 106 106 LC R 108 108 108 LC R 142142 142 LC K 149 149 149

In some other aspects, a THIOMAB™ antibody comprises one of the heavy orlight chain cysteine substitutions listed in Table 4.

TABLE 4 Screening GNE Kabat Chain Mutation Mutation Mutation (HC/LC)Residue Site # Site # Site # LC K 149 149 149 HC A 143 140 136 HC A 121118 114

Cysteine engineered antibodies which may be useful in the antibody-drugconjugates of the invention in the treatment of cancer include, but arenot limited to, antibodies against cell surface receptors andtumor-associated antigens (TAA). Tumor-associated antigens are known inthe art, and can be prepared for use in generating antibodies usingmethods and information which are well known in the art. In attempts todiscover effective cellular targets for cancer diagnosis and therapy,researchers have sought to identify transmembrane or otherwisetumor-associated polypeptides that are specifically expressed on thesurface of one or more particular type(s) of cancer cell as compared toon one or more normal non-cancerous cell(s). Often, suchtumor-associated polypeptides are more abundantly expressed on thesurface of the cancer cells as compared to on the surface of thenon-cancerous cells. The identification of such tumor-associated cellsurface antigen polypeptides has given rise to the ability tospecifically target cancer cells for destruction via antibody-basedtherapies.

Examples of tumor-associated antigens TAA include, but are not limitedto, TAA (1)-(53) listed herein. For convenience, information relating tothese antigens, all of which are known in the art, is listed herein andincludes names, alternative names, Genbank accession numbers and primaryreference(s), following nucleic acid and protein sequence identificationconventions of the National Center for Biotechnology Information (NCBI).Nucleic acid and protein sequences corresponding to TAA (1)-(53) areavailable in public databases such as GenBank. Tumor-associated antigenstargeted by antibodies include all amino acid sequence variants andisoforms possessing at least about 70%, 80%, 85%, 90%, or 95% sequenceidentity relative to the sequences identified in the cited references,or which exhibit substantially the same biological properties orcharacteristics as a TAA having a sequence found in the citedreferences. For example, a TAA having a variant sequence generally isable to bind specifically to an antibody that binds specifically to theTAA with the corresponding sequence listed. The sequences and disclosurein the reference specifically recited herein are expressly incorporatedby reference.

Tumor-Associated Antigens

(1) BMPR1B (bone morphogenetic protein receptor-type IB, Genbankaccession no. NM_001203) ten Dijke, P., et al. Science 264(5155):101-104 (1994), Oncogene 14 (11):1377-1382 (1997)); WO2004063362(Claim 2); WO2003042661 (Claim 12); U52003134790-A1 (Page 38-39);WO2002102235 (Claim 13; Page 296); WO2003055443 (Page 91-92);WO200299122 (Example 2; Page 528-530); WO2003029421 (Claim 6);WO2003024392 (Claim 2; FIG. 112); WO200298358 (Claim 1; Page 183);WO200254940 (Page 100-101); WO200259377(Page 349-350); WO200230268(Claim 27; Page 376); WO200148204 (Example; FIG. 4) NP_001194 bonemorphogenetic protein receptor, typeIB/pid=NP_001194.1—Cross-references: MIM:603248; NP_001194.1; AY065994.

(2) E16 (LAT1, SLC7A5, Genbank accession no. NM_003486) Biochem.Biophys. Res. Commun. 255 (2), 283-288 (1999), Nature 395 (6699):288-291(1998), Gaugitsch, H. W., et al. (1992) J. Biol. Chem. 267(16):11267-11273); WO2004048938 (Example 2); WO2004032842 (Example IV);WO2003042661 (Claim 12); WO2003016475 (Claim 1); WO200278524 (Example2); WO200299074 (Claim 19; Page 127-129); WO200286443 (Claim 27; Pages222, 393); WO2003003906 (Claim 10; Page 293); WO200264798 (Claim 33;Page 93-95); WO200014228 (Claim 5; Page 133-136); US2003224454 (FIG. 3);WO2003025138 (Claim 12; Page 150); NP_003477 solute carrier family 7(cationic amino acid transporter, y+system), member5/pid=NP_003477.3—Homo sapiens Cross-references: MIM:600182;NP_003477.3; NM_015923; NM_003486_1.

(3) STEAP1 (six transmembrane epithelial antigen of prostate, Genbankaccession no. NM_012449) Cancer Res. 61 (15), 5857-5860 (2001), Hubert,R. S., et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96 (25):14523-14528);WO2004065577 (Claim 6); WO2004027049 (FIG. 1L); EP1394274 (Example 11);WO2004016225 (Claim 2); WO2003042661 (Claim 12); US2003157089 (Example5); US2003185830 (Example 5); US2003064397 (FIG. 2); WO200289747(Example 5; Page 618-619); WO2003022995 (Example 9; FIG. 13A, Example53; Page 173, Example 2; FIG. 2A); NP_036581 six transmembraneepithelial antigen of the prostate Cross-references: MIM:604415;NP_036581.1; NM_012449_1.

(4) 0772P (CA125, MUC16, Genbank accession no. AF361486) J. Biol. Chem.276 (29):27371-27375 (2001)); WO2004045553 (Claim 14); WO200292836(Claim 6; FIG. 12); WO200283866 (Claim 15; Page 116-121); US2003124140(Example 16); U.S. Pat. No. 798,959. Cross-references: GI:34501467;AAK74120.3; AF361486_1.

(5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin,Genbank accession no. NM_005823) Yamaguchi, N., et al. Biol. Chem. 269(2), 805-808 (1994), Proc. Natl. Acad. Sci. U.S.A. 96 (20):11531-11536(1999), Proc. Natl. Acad. Sci. U.S.A. 93 (1):136-140 (1996), J. Biol.Chem. 270 (37):21984-21990 (1995)); WO2003101283 (Claim 14);(WO2002102235 (Claim 13; Page 287-288); WO2002101075 (Claim 4; Page308-309); WO200271928 (Page 320-321); WO9410312 (Page 52-57);Cross-references: MIM:601051; NP_005814.2; NM_005823_1.

(6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodiumphosphate), member 2, type II sodium-dependent phosphate transporter 3b,Genbank accession no. NM_006424) J. Biol. Chem. 277 (22):19665-19672(2002), Genomics 62 (2):281-284 (1999), J. A., et al. (1999) Biochem.Biophys. Res. Commun. 258 (3):578-582); WO2004022778 (Claim 2);EP1394274 (Example 11); WO2002102235 (Claim 13; Page 326); EP875569(Claim 1; Page 17-19); WO200157188 (Claim 20; Page 329); WO2004032842(Example IV); WO200175177 (Claim 24; Page 139-140); Cross-references:MIM:604217; NP_006415.1; NM_006424_1.

(7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMASB, SEMAG, Semaphorin 5bHlog, sema domain, seven thrombospondin repeats (type 1 and type1-like), transmembrane domain (TM) and short cytoplasmic domain,(semaphorin) 5B, Genbank accession no. AB040878) Nagase T., et al.(2000) DNA Res. 7 (2):143-150); WO2004000997 (Claim 1); WO2003003984(Claim 1); WO200206339 (Claim 1; Page 50); WO200188133 (Claim 1; Page41-43, 48-58); WO2003054152 (Claim 20); WO2003101400 (Claim 11);Accession: Q9P283; EMBL; AB040878; BAA95969.1. Genew; HGNC:10737.

(8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12, RIKENcDNA 2700050C12 gene, Genbank accession no. AY358628); Ross et al.(2002) Cancer Res. 62:2546-2553; US2003129192 (Claim 2); US2004044180(Claim 12); US2004044179 (Claim 11); US2003096961 (Claim 11);US2003232056 (Example 5); WO2003105758 (Claim 12); US2003206918 (Example5); EP1347046 (Claim 1); WO2003025148 (Claim 20); Cross-references:GI:37182378; AAQ88991.1; AY358628_1.

(9) ETBR (Endothelin type B receptor, Genbank accession no. AY275463);Nakamuta M., et al. Biochem. Biophys. Res. Commun. 177, 34-39, 1991;Ogawa Y., et al. Biochem. Biophys. Res. Commun. 178, 248-255, 1991; AraiH., et al. Jpn. Circ. J. 56, 1303-1307, 1992; Arai H., et al. J. Biol.Chem. 268, 3463-3470, 1993; Sakamoto A., Yanagisawa M., et al. Biochem.Biophys. Res. Commun. 178, 656-663, 1991; Elshourbagy N. A., et al. J.Biol. Chem. 268, 3873-3879, 1993; Haendler B., et al. J. Cardiovasc.Pharmacol. 20, s1-S4, 1992; Tsutsumi M., et al. Gene 228, 43-49, 1999;Strausberg R. L., et al. Proc. Natl. Acad. Sci. U.S.A. 99, 16899-16903,2002; Bourgeois C., et al. J. Clin. Endocrinol. Metab. 82, 3116-3123,1997; Okamoto Y., et al. Biol. Chem. 272, 21589-21596, 1997; Verheij J.B., et al. Am. J. Med. Genet. 108, 223-225, 2002; Hofstra R. M. W., etal. Eur. J. Hum. Genet. 5, 180-185, 1997; Puffenberger E. G., et al.Cell 79, 1257-1266, 1994; Attie T., et al., Hum. Mol. Genet. 4,2407-2409, 1995; Auricchio A., et al. Hum. Mol. Genet. 5:351-354, 1996;Amiel J., et al. Hum. Mol. Genet. 5, 355-357, 1996; Hofstra R. M. W., etal. Nat. Genet. 12, 445-447, 1996; Svensson P. J., et al. Hum. Genet.103, 145-148, 1998; Fuchs S., et al. Mol. Med. 7, 115-124, 2001;Pingault V., et al. (2002) Hum. Genet. 111, 198-206; WO2004045516 (Claim1); WO2004048938 (Example 2); WO2004040000 (Claim 151); WO2003087768(Claim 1); WO2003016475 (Claim 1); WO2003016475 (Claim 1); WO200261087(FIG. 1); WO2003016494 (FIG. 6); WO2003025138 (Claim 12; Page 144);WO200198351 (Claim 1; Page 124-125); EP522868 (Claim 8; FIG. 2);WO200177172 (Claim 1; Page 297-299); US2003109676; U.S. Pat. No.6,518,404 (FIG. 3); U.S. Pat. No. 5,773,223 (Claim 1a; Col 31-34);WO2004001004.

(10) MSG783 (RNF124, hypothetical protein F1120315, Genbank accessionno. NM_017763); WO2003104275 (Claim 1); WO2004046342 (Example 2);WO2003042661 (Claim 12); WO2003083074 (Claim 14; Page 61); WO2003018621(Claim 1); WO2003024392 (Claim 2; FIG. 93); WO200166689 (Example 6);Cross-references: LocusID:54894; NP_060233.2; NM_017763_1.

(11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostatecancer associated gene 1, prostate cancer associated protein 1, sixtransmembrane epithelial antigen of prostate 2, six transmembraneprostate protein, Genbank accession no. AF455138) Lab. Invest. 82(11):1573-1582 (2002)); WO2003087306; US2003064397 (Claim 1; FIG. 1);WO200272596 (Claim 13; Page 54-55); WO200172962 (Claim 1; FIG. 4B);WO2003104270 (Claim 11); WO2003104270 (Claim 16); US2004005598 (Claim22); WO2003042661 (Claim 12); US2003060612 (Claim 12; FIG. 10);WO200226822 (Claim 23; FIG. 2); WO200216429 (Claim 12; FIG. 10);Cross-references: GI:22655488; AAN04080.1; AF455138_1.

(12) TrpM4 (BR22450, F1120041, TRPM4, TRPM4B, transient receptorpotential cation channel, subfamily M, member 4, Genbank accession no.NM_017636) Xu, X. Z., et al. Proc. Natl. Acad. Sci. U.S.A. 98(19):10692-10697 (2001), Cell 109 (3):397-407 (2002), J. Biol. Chem. 278(33):30813-30820 (2003)); US2003143557 (Claim 4); WO200040614 (Claim 14;Page 100-103); WO200210382 (Claim 1; FIG. 9A); WO2003042661 (Claim 12);WO200230268 (Claim 27; Page 391); US2003219806 (Claim 4); WO200162794(Claim 14; FIG. 1A-D); Cross-references: MIM:606936; NP_060106.2;NM_017636_1.

(13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derivedgrowth factor, Genbank accession no. NP_003203 or NM_003212)Ciccodicola, A., et al. EMBO J. 8 (7):1987-1991 (1989), Am. J. Hum.Genet. 49 (3):555-565 (1991)); US2003224411 (Claim 1); WO2003083041(Example 1); WO2003034984 (Claim 12); WO200288170 (Claim 2; Page 52-53);WO2003024392 (Claim 2; FIG. 58); WO200216413 (Claim 1; Page 94-95, 105);WO200222808 (Claim 2; FIG. 1); U.S. Pat. No. 5,854,399 (Example 2; Col17-18); U.S. Pat. No. 5,792,616 (FIG. 2); Cross-references: MIM:187395;NP_003203.1; NM_003212_1.

(14) CD21 (CR2 (Complement receptor 2) or C3DR (C3d/Epstein Barr virusreceptor) or Hs.73792 Genbank accession no. M26004) Fujisaku et al.(1989) J. Biol. Chem. 264 (4):2118-2125); Weis J. J., et al. J. Exp.Med. 167, 1047-1066, 1988; Moore M., et al. Proc. Natl. Acad. Sci.U.S.A. 84, 9194-9198, 1987; Barel M., et al. Mol. Immunol. 35,1025-1031, 1998; Weis J. J., et al. Proc. Natl. Acad. Sci. U.S.A. 83,5639-5643, 1986; Sinha S. K., et al. (1993) J. Immunol. 150, 5311-5320;WO2004045520 (Example 4); US2004005538 (Example 1); WO2003062401 (Claim9); WO2004045520 (Example 4); WO9102536 (FIGS. 9.1-9.9); WO2004020595(Claim 1); Accession: P20023; Q13866; Q14212; EMBL; M26004; AAA35786.1.

(15) CD79b (CD79B, CD79β, IGb (immunoglobulin-associated beta), B29,Genbank accession no. NM_000626 or 11038674) Proc. Natl. Acad. Sci.U.S.A. (2003) 100 (7):4126-4131, Blood (2002) 100 (9):3068-3076, Mulleret al. (1992) Eur. J. Immunol. 22 (6):1621-1625); WO2004016225 (Claim 2,FIG. 140); WO2003087768, US2004101874 (Claim 1, page 102); WO2003062401(Claim 9); WO200278524 (Example 2); US2002150573 (Claim 5, page 15);U.S. Pat. No. 5,644,033; WO2003048202 (Claim 1, pages 306 and 309); WO99/558658, U.S. Pat. No. 6,534,482 (Claim 13, FIG. 17A/B); WO200055351(Claim 11, pages 1145-1146); Cross-references: MIM:147245; NP_000617.1;NM_000626_1.

(16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphataseanchor protein 1a), SPAP1B, SPAP1C, Genbank accession no. NM_030764,AY358130) Genome Res. 13 (10):2265-2270 (2003), Immunogenetics 54(2):87-95 (2002), Blood 99 (8):2662-2669 (2002), Proc. Natl. Acad. Sci.U.S.A. 98 (17):9772-9777 (2001), Xu, M. J., et al. (2001) Biochem.Biophys. Res. Commun. 280 (3):768-775; WO2004016225 (Claim 2);WO2003077836; WO200138490 (Claim 5; FIG. 18D-1-18D-2); WO2003097803(Claim 12); WO2003089624 (Claim 25); Cross-references: MIM:606509;NP_110391.2; NM_030764_1.

(17) HER2 (ErbB2, Genbank accession no. M11730) Coussens L., et al.Science (1985) 230(4730):1132-1139); Yamamoto T., et al. Nature 319,230-234, 1986; Semba K., et al. Proc. Natl. Acad. Sci. U.S.A. 82,6497-6501, 1985; Swiercz J. M., et al. J. Cell Biol. 165, 869-880, 2004;Kuhns J. J., et al. J. Biol. Chem. 274, 36422-36427, 1999; Cho H.-S., etal. Nature 421, 756-760, 2003; Ehsani A., et al. (1993) Genomics 15,426-429; WO2004048938 (Example 2); WO2004027049 (FIG. 10; WO2004009622;WO2003081210; WO2003089904 (Claim 9); WO2003016475 (Claim 1);US2003118592; WO2003008537 (Claim 1); WO2003055439 (Claim 29; FIG.1A-B); WO2003025228 (Claim 37; FIG. 5C); WO200222636 (Example 13; Page95-107); WO200212341 (Claim 68; FIG. 7); WO200213847 (Page 71-74);WO200214503 (Page 114-117); WO200153463 (Claim 2; Page 41-46);WO200141787 (Page 15); WO200044899 (Claim 52; FIG. 7); WO200020579(Claim 3; FIG. 2); U.S. Pat. No. 5,869,445 (Claim 3; Col 31-38);WO9630514 (Claim 2; Page 56-61); EP1439393 (Claim 7); WO2004043361(Claim 7); WO2004022709; WO200100244 (Example 3; FIG. 4); Accession:P04626; EMBL; M11767; AAA35808.1. EMBL; M11761; AAA35808.1.

(18) NCA (CEACAM6, Genbank accession no. M18728); Barnett T., et al.Genomics 3, 59-66, 1988; Tawaragi Y., et al. Biochem. Biophys. Res.Commun. 150, 89-96, 1988; Strausberg R. L., et al. Proc. Natl. Acad.Sci. U.S.A. 99:16899-16903, 2002; WO2004063709; EP1439393 (Claim 7);WO2004044178 (Example 4); WO2004031238; WO2003042661 (Claim 12);WO200278524 (Example 2); WO200286443 (Claim 27; Page 427); WO200260317(Claim 2); Accession: P40199; Q14920; EMBL; M29541; AAA59915.1. EMBL;M18728.

(19) MDP (DPEP1, Genbank accession no. BC017023) Proc. Natl. Acad. Sci.U.S.A. 99 (26):16899-16903 (2002)); WO2003016475 (Claim 1); WO200264798(Claim 33; Page 85-87); JP05003790 (FIG. 6-8); WO9946284 (FIG. 9);Cross-references: MIM:179780; AAH17023.1; BC017023_1.

(20) IL20Rα (IL20Rα, ZCYTOR7, Genbank accession no. AF184971); Clark H.F., et al. Genome Res. 13, 2265-2270, 2003; Mungall A. J., et al. Nature425, 805-811, 2003; Blumberg H., et al. Cell 104, 9-19, 2001; DumoutierL., et al. J. Immunol. 167, 3545-3549, 2001; Parrish-Novak J., et al. J.Biol. Chem. 277, 47517-47523, 2002; Pletnev S., et al. (2003)Biochemistry 42:12617-12624; Sheikh F., et al. (2004) J. Immunol. 172,2006-2010; EP1394274 (Example 11); US2004005320 (Example 5);WO2003029262 (Page 74-75); WO2003002717 (Claim 2; Page 63); WO200222153(Page 45-47); US2002042366 (Page 20-21); WO200146261 (Page 57-59);WO200146232 (Page 63-65); WO9837193 (Claim 1; Page 55-59); Accession:Q9UHF4; Q6UWA9; Q96SH8; EMBL; AF184971; AAF01320.1.

(21) Brevican (BCAN, BEHAB, Genbank accession no. AF229053) Gary S. C.,et al. Gene 256, 139-147, 2000; Clark H. F., et al. Genome Res. 13,2265-2270, 2003; Strausberg R. L., et al. Proc. Natl. Acad. Sci. U.S.A.99, 16899-16903, 2002; US2003186372 (Claim 11); US2003186373 (Claim 11);US2003119131 (Claim 1; FIG. 52); US2003119122 (Claim 1; FIG. 52);US2003119126 (Claim 1); US2003119121 (Claim 1; FIG. 52); US2003119129(Claim 1); US2003119130 (Claim 1); US2003119128 (Claim 1; FIG. 52);US2003119125 (Claim 1); WO2003016475 (Claim 1); WO200202634 (Claim 1).

(22) EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5, Genbank accession no.NM_004442) Chan, J. and Watt, V. M., Oncogene 6 (6), 1057-1061 (1991)Oncogene 10 (5):897-905 (1995), Annu. Rev. Neurosci. 21:309-345 (1998),Int. Rev. Cytol. 196:177-244 (2000)); WO2003042661 (Claim 12);WO200053216 (Claim 1; Page 41); WO2004065576 (Claim 1); WO2004020583(Claim 9); WO2003004529 (Page 128-132); WO200053216 (Claim 1; Page 42);Cross-references: MIM:600997; NP_004433.2; NM_004442_1.

(23) ASLG659 (B7h, Genbank accession no. AX092328) US20040101899 (Claim2); WO2003104399 (Claim 11); WO2004000221 (FIG. 3); US2003165504 (Claim1); US2003124140 (Example 2); US2003065143 (FIG. 60); WO2002102235(Claim 13; Page 299); US2003091580 (Example 2); WO200210187 (Claim 6;FIG. 10); WO200194641 (Claim 12; FIG. 7b); WO200202624 (Claim 13; FIG.1A-1B); US2002034749 (Claim 54; Page 45-46); WO200206317 (Example 2;Page 320-321, Claim 34; Page 321-322); WO200271928 (Page 468-469);WO200202587 (Example 1; FIG. 1); WO200140269 (Example 3; Pages 190-192);WO200036107 (Example 2; Page 205-207); WO2004053079 (Claim 12);WO2003004989 (Claim 1); WO200271928 (Page 233-234, 452-453); WO 0116318.

(24) PSCA (Prostate stem cell antigen precursor, Genbank accession no.AJ297436) Reiter R. E., et al. Proc. Natl. Acad. Sci. U.S.A. 95,1735-1740, 1998; Gu Z., et al. Oncogene 19, 1288-1296, 2000; Biochem.Biophys. Res. Commun. (2000) 275(3):783-788; WO2004022709; EP1394274(Example 11); US2004018553 (Claim 17); WO2003008537 (Claim 1);WO200281646 (Claim 1; Page 164); WO2003003906 (Claim 10; Page 288);WO200140309 (Example 1; FIG. 17); US2001055751 (Example 1; FIG. 1b);WO200032752 (Claim 18; FIG. 1); WO9851805 (Claim 17; Page 97); WO9851824(Claim 10; Page 94); WO9840403 (Claim 2; FIG. 1B); Accession: 043653;EMBL; AF043498; AAC39607.1.

(25) GEDA (Genbank accession No. AY260763); AAP14954 lipoma HMGICfusion-partner-like protein/pid=AAP14954.1—Homo sapiens Species: Homosapiens (human) WO2003054152 (Claim 20); WO2003000842 (Claim 1);WO2003023013 (Example 3, Claim 20); US2003194704 (Claim 45);Cross-references: GI:30102449; AAP14954.1; AY260763_1.

(26) BAFF-R (B cell-activating factor receptor, BLyS receptor 3, BR3,Genbank accession No. AF116456); BAFF receptor/pid=NP_443177.1—Homosapiens Thompson, J. S., et al. Science 293 (5537), 2108-2111 (2001);WO2004058309; WO2004011611; WO2003045422 (Example; Page 32-33);WO2003014294 (Claim 35; FIG. 6B); WO2003035846 (Claim 70; Page 615-616);WO200294852 (Col 136-137); WO200238766 (Claim 3; Page 133); WO200224909(Example 3; FIG. 3); Cross-references: MIM:606269; NP_443177.1;NM_052945_1; AF 132600.

(27) CD22 (B-cell receptor CD22-B isoform, BL-CAM, Lyb-8, Lyb8,SIGLEC-2, F1122814, Genbank accession No. AK026467); Wilson et al.(1991) J. Exp. Med. 173:137-146; WO2003072036 (Claim 1; FIG. 1);Cross-references: MIM:107266; NP_001762.1; NM_001771_1.

(28) CD79a (CD79A, CD79α, immunoglobulin-associated alpha, a Bcell-specific protein that covalently interacts with Ig beta (CD79B) andforms a complex on the surface with Ig M molecules, transduces a signalinvolved in B-cell differentiation), pI: 4.84, MW: 25028 TM: 2 [P] GeneChromosome: 19q13.2, Genbank accession No. NP_001774.10) WO2003088808,US20030228319; WO2003062401 (Claim 9); US2002150573 (Claim 4, pages13-14); WO9958658 (Claim 13, FIG. 16); WO9207574 (FIG. 1); U.S. Pat. No.5,644,033; Ha et al. (1992) J. Immunol. 148(5):1526-1531; Mueller et al.(1992) Eur. J. Biochem. 22:1621-1625; Hashimoto et al. (1994)Immunogenetics 40(4):287-295; Preud'homme et al. (1992) Clin. Exp.Immunol. 90(1):141-146; Yu et al. (1992) J. Immunol. 148(2) 633-637;Sakaguchi et al. (1988) EMBO J. 7(11):3457-3464.

(29) CXCR5 (Burkitt's lymphoma receptor 1, a G protein-coupled receptorthat is activated by the CXCL13 chemokine, functions in lymphocytemigration and humoral defense, plays a role in HIV-2 infection andperhaps development of AIDS, lymphoma, myeloma, and leukemia); 372 aa,pI: 8.54 MW: 41959 TM: 7 [P] Gene Chromosome: 11q23.3, Genbank accessionNo. NP_001707.1) WO2004040000; WO2004015426; US2003105292 (Example 2);U.S. Pat. No. 6,555,339 (Example 2); WO200261087 (FIG. 1); WO200157188(Claim 20, page 269); WO200172830 (pages 12-13); WO200022129 (Example 1,pages 152-153, Example 2, pages 254-256); WO9928468 (Claim 1, page 38);U.S. Pat. No. 5,440,021 (Example 2, col 49-52); WO9428931 (pages 56-58);WO9217497 (Claim 7, FIG. 5); Dobner et al. (1992) Eur. J. Immunol.22:2795-2799; Barella et al. (1995) Biochem. J. 309:773-779.

(30) HLA-DOB (Beta subunit of MHC class II molecule (Ia antigen) thatbinds peptides and presents them to CD4+T lymphocytes); 273 aa, pI: 6.56MW: 30820 TM: 1 [P] Gene Chromosome: 6p21.3, Genbank accession No.NP_002111.1) Tonnelle et al. (1985) EMBO J. 4(11):2839-2847; Jonsson etal. (1989) Immunogenetics 29(6):411-413; Beck et al. (1992) J. Mol.Biol. 228:433-441; Strausberg et al. (2002) Proc. Natl. Acad. Sci USA99:16899-16903; Servenius et al. (1987) J. Biol. Chem. 262:8759-8766;Beck et al. (1996) J. Mol. Biol. 255:1-13; Naruse et al. (2002) TissueAntigens 59:512-519; WO9958658 (Claim 13, FIG. 15); U.S. Pat. No.6,153,408 (Col 35-38); U.S. Pat. No. 5,976,551 (col 168-170); U.S. Pat.No. 6,011,146 (col 145-146); Kasahara et al. (1989) Immunogenetics30(1):66-68; Larhammar et al. (1985) J. Biol. Chem. 260(26):14111-14119.

(31) P2X5 (Purinergic receptor P2X ligand-gated ion channel 5, an ionchannel gated by extracellular ATP, may be involved in synaptictransmission and neurogenesis, deficiency may contribute to thepathophysiology of idiopathic detrusor instability); 422 aa), pI: 7.63,MW: 47206 TM: 1 [P] Gene Chromosome: 17p13.3, Genbank accession No.NP_002552.2) Le et al. (1997) FEBS Lett. 418(1-2):195-199; WO2004047749;WO2003072035 (Claim 10); Touchman et al. (2000) Genome Res. 10:165-173;WO200222660 (Claim 20); WO2003093444 (Claim 1); WO2003087768 (Claim 1);WO2003029277 (page 82).

(32) CD72 (B-cell differentiation antigen CD72, Lyb-2) PROTEIN SEQUENCEFull maeaity . . . tafrfpd (1.359; 359 aa), pI: 8.66, MW: 40225 TM: 1[P] Gene Chromosome: 9p13.3, Genbank accession No. NP_001773.1)WO2004042346 (Claim 65); WO2003026493 (pages 51-52, 57-58); WO200075655(pages 105-106); Von Hoegen et al. (1990) J. Immunol. 144(12):4870-4877;Strausberg et al. (2002) Proc. Natl. Acad. Sci USA 99:16899-16903.

(33) LY64 (Lymphocyte antigen 64 (RP105), type I membrane protein of theleucine rich repeat (LRR) family, regulates B-cell activation andapoptosis, loss of function is associated with increased diseaseactivity in patients with systemic lupus erythematosis); 661 aa, pI:6.20, MW: 74147 TM: 1 [P] Gene Chromosome: 5q12, Genbank accession No.NP_005573.1) US2002193567; WO9707198 (Claim 11, pages 39-42); Miura etal. (1996) Genomics 38(3):299-304; Miura et al. (1998) Blood92:2815-2822; WO2003083047; WO9744452 (Claim 8, pages 57-61);WO200012130 (pages 24-26).

(34) FcRH1 (Fc receptor-like protein 1, a putative receptor for theimmunoglobulin Fc domain that contains C2 type Ig-like and ITAM domains,may have a role in B-lymphocyte differentiation); 429 aa, pI: 5.28, MW:46925 TM: 1 [P] Gene Chromosome: 1q21-1q22, Genbank accession No.NP_443170.1) WO2003077836; WO200138490 (Claim 6, FIG. 18E-1-18-E-2);Davis et al. (2001) Proc. Natl. Acad. Sci USA 98(17):9772-9777;WO2003089624 (Claim 8); EP1347046 (Claim 1); WO2003089624 (Claim 7).

(35) IRTA2 (Immunoglobulin superfamily receptor translocation associated2, a putative immunoreceptor with possible roles in B cell developmentand lymphomagenesis; deregulation of the gene by translocation occurs insome B cell malignancies); 977 aa, pI: 6.88 MW: 106468 TM: 1 [P] GeneChromosome: 1q21, Genbank accession No. Human:AF343662, AF343663,AF343664, AF343665, AF369794, AF397453, AK090423, AK090475, AL834187,AY358085; Mouse:AK089756, AY158090, AY506558; NP_112571.1. WO2003024392(Claim 2, FIG. 97); Nakayama et al. (2000) Biochem. Biophys. Res.Commun. 277(1):124-127; WO2003077836; WO200138490 (Claim 3, FIG.18B-1-18B-2).

(36) TENB2 (TMEFF2, tomoregulin, TPEF, HPP1, TR, putative transmembraneproteoglycan, related to the EGF/heregulin family of growth factors andfollistatin); 374 aa, NCBI Accession: AAD55776, AAF91397, AAG49451, NCBIRefSeq: NP_057276; NCBI Gene: 23671; OMIM: 605734; SwissProt Q9UIK5;Genbank accession No. AF179274; AY358907, CAF85723, CQ782436WO2004074320 (SEQ ID NO 810); JP2004113151 (SEQ ID NOS 2, 4, 8);WO2003042661 (SEQ ID NO 580); WO2003009814 (SEQ ID NO 411); EP1295944(pages 69-70); WO200230268 (page 329); WO200190304 (SEQ ID NO 2706);US2004249130; US2004022727; WO2004063355; US2004197325; US2003232350;US2004005563; US2003124579; Horie et al. (2000) Genomics 67:146-152;Uchida et al. (1999) Biochem. Biophys. Res. Commun. 266:593-602; Lianget al. (2000) Cancer Res. 60:4907-12; Glynne-Jones et al. (2001) Int JCancer. October 15; 94(2):178-84.

(37) PMEL17 (silver homologs SILV; D12S53E; PMEL17; SI; SIL); ME20;gp100) BC001414; BT007202; M32295; M77348; NM_006928; McGlinchey, R. P.et al. (2009) Proc. Natl. Acad. Sci. U.S.A. 106 (33), 13731-13736;Kummer, M. P. et al. (2009) J. Biol. Chem. 284 (4), 2296-2306.

(38) TMEFF1 (transmembrane protein with EGF-like and twofollistatin-like domains I; Tomoregulin-1); H7365; C9orf2; C9ORF2;U19878; X83961; NM_080655; NM_003692; Harms, P. W. (2003) Genes Dev. 17(21), 2624-2629; Gery, S. et al. (2003) Oncogene 22 (18):2723-2727.

(39) GDNF-Ra1 (GDNF family receptor alpha 1; GFRA1; GDNFR; GDNFRA;RETL1; TRNR1; RET1L; GDNFR-alpha1; GFR-ALPHA-1); U95847; BC014962;NM_145793 NM_005264; Kim, M. H. et al. (2009) Mol. Cell. Biol. 29 (8),2264-2277; Treanor, J. J. et al. (1996) Nature 382 (6586):80-83.

(40) Ly6E (lymphocyte antigen 6 complex, locus E; Ly67, RIG-E, SCA-2,TSA-1); NP_002337.1; NM_002346.2; de Nooij-van Dalen, A. G. et al.(2003) Int. J. Cancer 103 (6), 768-774; Zammit, D. J. et al. (2002) Mol.Cell. Biol. 22 (3):946-952.

(41) TMEM46 (shisa homolog 2 (Xenopus laevis); SHISA2); NP_001007539.1;NM_001007538.1; Furushima, K. et al. (2007) Dev. Biol. 306 (2), 480-492;Clark, H. F. et al. (2003) Genome Res. 13 (10):2265-2270.

(42) Ly6G6D (lymphocyte antigen 6 complex, locus G6D; Ly6-D, MEGT1);NP_067079.2; NM_021246.2; Mallya, M. et al. (2002) Genomics 80(1):113-123; Ribas, G. et al. (1999) J. Immunol. 163 (1):278-287.

(43) LGR5 (leucine-rich repeat-containing G protein-coupled receptor 5;GPR49, GPR67); NP_003658.1; NM_003667.2; Salanti, G. et al. (2009) Am.J. Epidemiol. 170 (5):537-545; Yamamoto, Y. et al. (2003) Hepatology 37(3):528-533.

(44) RET (ret proto-oncogene; MEN2A; HSCR1; MEN2B; MTC1; PTC; CDHF12;Hs.168114; RET51; RET-ELE1); NP_066124.1; NM_020975.4; Tsukamoto, H. etal. (2009) Cancer Sci. 100 (10):1895-1901; Narita, N. et al. (2009)Oncogene 28 (34):3058-3068.

(45) LY6K (lymphocyte antigen 6 complex, locus K; LY6K; HSJ001348;FLJ35226); NP_059997.3; NM_017527.3; Ishikawa, N. et al. (2007) CancerRes. 67 (24):11601-11611; de Nooij-van Dalen, A. G. et al. (2003) Int.J. Cancer 103 (6):768-774.

(46) GPR19 (G protein-coupled receptor 19; Mm.4787); NP_006134.1;NM_006143.2; Montpetit, A. and Sinnett, D. (1999) Hum. Genet. 105(1-2):162-164; O'Dowd, B. F. et al. (1996) FEBS Lett. 394 (3):325-329.

(47) GPR54 (KISS1 receptor, KISS1R; GPR54; HOT7T175; AXOR12);NP_115940.2; NM_032551.4; Navenot, J. M. et al. (2009) Mol. Pharmacol.75 (6):1300-1306; Hata, K. et al. (2009) Anticancer Res. 29 (2):617-623.

(48) ASPHD1 (aspartate beta-hydroxylase domain containing 1; LOC253982);NP_859069.2; NM_181718.3; Gerhard, D. S. et al. (2004) Genome Res. 14(10B):2121-2127.

(49) Tyrosinase (TYR; OCAIA; OCA1A; tyrosinase; SHEP3); NP_000363.1;NM_000372.4; Bishop, D. T. et al. (2009) Nat. Genet. 41 (8):920-925;Nan, H. et al. (2009) Int. J. Cancer 125 (4):909-917.

(50) TMEM118 (ring finger protein, transmembrane 2; RNFT2; FLJ14627);NP_001103373.1; NM_001109903.1; Clark, H. F. et al. (2003) Genome Res.13 (10):2265-2270; Scherer, S. E. et al. (2006) Nature 440(7082):346-351.

(51) GPR172A (G protein-coupled receptor 172A; GPCR41; FLJ11856;D15Ertd747e); NP_078807.1; NM_024531.3; Ericsson, T. A. et al. (2003)Proc. Natl. Acad. Sci. U.S.A. 100 (11):6759-6764; Takeda, S. et al.(2002) FEBS Lett. 520 (1-3):97-101.

(52) CD33, a member of the sialic acid binding, immunoglobulin-likelectin family, is a 67-kDa glycosylated transmembrane protein. CD33 isexpressed on most myeloid and monocytic leukemia cells in addition tocommitted myelomonocytic and erythroid progenitor cells. It is not seenon the earliest pluripotent stem cells, mature granulocytes, lymphoidcells, or nonhematopoietic cells (Sabbath et al., (1985) J. Clin.Invest. 75:756-56; Andrews et al., (1986) Blood 68:1030-5). CD33contains two tyrosine residues on its cytoplasmic tail, each of which isfollowed by hydrophobic residues similar to the immunoreceptortyrosine-based inhibitory motif (ITIM) seen in many inhibitoryreceptors.

(53) CLL-1 (CLEC12A, MICL, and DCAL2), encodes a member of the C-typelectin/C-type lectin-like domain (CTL/CTLD) superfamily. Members of thisfamily share a common protein fold and have diverse functions, such ascell adhesion, cell-cell signaling, glycoprotein turnover, and roles ininflammation and immune response. The protein encoded by this gene is anegative regulator of granulocyte and monocyte function. Severalalternatively spliced transcript variants of this gene have beendescribed, but the full-length nature of some of these variants has notbeen determined. This gene is closely linked to other CTL/CTLDsuperfamily members in the natural killer gene complex region onchromosome 12p13 (Drickamer K (1999) Curr. Opin. Struct. Biol. 9(5):585-90; van Rhenen A, et al., (2007) Blood 110 (7):2659-66; Chen CH, et al. (2006) Blood 107 (4):1459-67; Marshall A S, et al. (2006) Eur.J. Immunol. 36 (8):2159-69; Bakker A B, et al. (2005) Cancer Res. 64(22):8443-50; Marshall A S, et al. (2004) J. Biol. Chem. 279(15):14792-802). CLL-1 has been shown to be a type II transmembranereceptor comprising a single C-type lectin-like domain (which is notpredicted to bind either calcium or sugar), a stalk region, atransmembrane domain and a short cytoplasmic tail containing an ITIMmotif.

Antibody Derivatives

In certain embodiments, an antibody provided herein may be furthermodified to contain additional nonproteinaceous moieties that are knownin the art and readily available. The moieties suitable forderivatization of the antibody include but are not limited to watersoluble polymers. Non-limiting examples of water soluble polymersinclude, but are not limited to, polyethylene glycol (PEG), copolymersof ethylene glycol/propylene glycol, carboxymethylcellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,polypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols(e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethyleneglycol propionaldehyde may have advantages in manufacturing due to itsstability in water. The polymer may be of any molecular weight, and maybe branched or unbranched. The number of polymers attached to theantibody may vary, and if more than one polymer is attached, they can bethe same or different molecules. In general, the number and/or type ofpolymers used for derivatization can be determined based onconsiderations including, but not limited to, the particular propertiesor functions of the antibody to be improved, whether the antibodyderivative will be used in a therapy under defined conditions, etc.

In another embodiment, conjugates of an antibody and nonproteinaceousmoiety that may be selectively heated by exposure to radiation areprovided. In one embodiment, the nonproteinaceous moiety is a carbonnanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605(2005)). The radiation may be of any wavelength, and includes, but isnot limited to, wavelengths that do not harm ordinary cells, but whichheat the nonproteinaceous moiety to a temperature at which cellsproximal to the antibody-nonproteinaceous moiety are killed.

Antibodies may be produced using recombinant methods and compositions,e.g., as described in U.S. Pat. No. 4,816,567. Such nucleic acid mayencode an amino acid sequence comprising the VL and/or an amino acidsequence comprising the VH of the antibody (e.g., the light and/or heavychains of the antibody). In a further embodiment, one or more vectors(e.g., expression vectors) comprising such nucleic acid are provided. Ina further embodiment, a host cell comprising such nucleic acid isprovided. In one such embodiment, a host cell comprises (e.g., has beentransformed with): (1) a vector comprising a nucleic acid that encodesan amino acid sequence comprising the VL of the antibody and an aminoacid sequence comprising the VH of the antibody, or (2) a first vectorcomprising a nucleic acid that encodes an amino acid sequence comprisingthe VL of the antibody and a second vector comprising a nucleic acidthat encodes an amino acid sequence comprising the VH of the antibody.In one embodiment, the host cell is eukaryotic, e.g. a Chinese HamsterOvary (CHO) cell or lymphoid cell (e.g., Y0, NS0, Sp20 cell).

For recombinant production of an antibody, nucleic acid encoding anantibody, e.g., as described herein, is isolated and inserted into oneor more vectors for further cloning and/or expression in a host cell.Such nucleic acid may be readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the antibody).

Suitable host cells for cloning or expression of antibody-encodingvectors include prokaryotic or eukaryotic cells described herein. Forexample, antibodies may be produced in bacteria, in particular whenglycosylation and Fc effector function are not needed. For expression ofantibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat.Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods inMolecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa,N.J., 2003), pp. 245-254, describing expression of antibody fragments inE. coli.) After expression, the antibody may be isolated from thebacterial cell paste in a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors, including fungi and yeast strains whoseglycosylation pathways have been “humanized,” resulting in theproduction of an antibody with a partially or fully human glycosylationpattern. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li etal., Nat. Biotech. 24:210-215 (2006).

Suitable host cells for the expression of glycosylated antibody are alsoderived from multicellular organisms (invertebrates and vertebrates).Examples of invertebrate cells include plant and insect cells. Numerousbaculoviral strains have been identified which may be used inconjunction with insect cells, particularly for transfection ofSpodoptera frugiperda cells.

Plant cell cultures can also be utilized as hosts. See, e.g., U.S. Pat.Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429(describing PLANTIBODIES™ technology for producing antibodies intransgenic plants).

Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977));baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells asdescribed, e.g., in Mather, Biol. Reprod. 23:243-251 (1980)); monkeykidney cells (CV1); African green monkey kidney cells (VERO-76); humancervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo ratliver cells (BRL 3A); human lung cells (W138); human liver cells (HepG2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., inMather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; andFS4 cells. Other useful mammalian host cell lines include Chinesehamster ovary (CHO) cells, including DHFR⁻CHO cells (Urlaub et al.,Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines suchas Y0, NS0 and Sp2/0. For a review of certain mammalian host cell linessuitable for antibody production, see, e.g., Yazaki and Wu, Methods inMolecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa,N.J.), pp. 255-268 (2003).

Antibody-Drug Conjugates

The invention provides antibody-drug conjugates having an antibodyherein conjugated to one or more calicheamicin derivative compounds.More particularly, the present disclosure provides an antibody-drugconjugate wherein the calicheamicin derivative compound is directlyconjugated to the antibody by means of a covalent bond, rather than themore conventional approach of a linker, a linker-spacer, alinker-reactive group, or the like.

Antibody-drug conjugates allow for the targeted delivery of a drugmoiety to a tumor, and, in some embodiments intracellular accumulationtherein, where systemic administration of unconjugated drugs may resultin unacceptable levels of toxicity to normal cells (Polakis P. (2005)Current Opinion in Pharmacology 5:382-387).

Antibody-drug conjugates are targeted chemotherapeutic molecules whichcombine properties of both antibodies and cytotoxic drugs by targetingpotent cytotoxic drugs to antigen-expressing tumor cells (Teicher, B. A.(2009) Current Cancer Drug Targets 9:982-1004), thereby enhancing thetherapeutic index by maximizing efficacy and minimizing off-targettoxicity (Carter, P. J. and Senter P. D. (2008) The Cancer Jour.14(3):154-169; Chari, R. V. (2008) Acc. Chem. Res. 41:98-107.

The antibody-drug conjugate compounds of the invention include thosewith anticancer activity. In some embodiments, the antibody-drugconjugate compounds include an antibody directly conjugated to thecalicheamicin drug moiety or derivative (i.e., the antibody is directlyattached or bound to the calicheamicin drug moiety or derivative, afterloss of a leaving group and without a linking group or moiety presentthere between). The antibody-drug conjugates of the inventionselectively deliver an effective dose of a drug to tumor tissue, wherebygreater selectivity (i.e., a lower efficacious dose) may be achievedwhile increasing the therapeutic index (“therapeutic window”).

As depicted below, an exemplary embodiment of an antibody-drug conjugatecompound comprises an antibody (Ab) which targets a tumor cell and acalicheamicin drug moiety (D) that is directly attached thereto by acovalent bond.

In such embodiments, R is suitably selected from H, —C(O)R¹, —C(O)NR¹R²,—S(O)₂R¹, and —S(O)₂NR²R¹. R¹ and R² may be independently selected fromhydrogen, optionally substituted C₁₋₆ alkyl and C₆₋₂₀ aryl. In someparticular aspects, R may be —C(O)CH₃. p refers to the equivalents ofcalicheamicin per Ab equivalent.

In some aspects Ab is an antibody which binds to one or moretumor-associated antigens or cell-surface receptors as describedelsewhere herein. In some other aspects, Ab is selected from BMPR1B,E16, STEAP1, MUC16, MPF, Napi2b, Sema 5b, PSCA hlg, ETBR, MSG783,STEAP2, TrpM4, CRIPTO, CD21, CD79b, FcRH2, HER2, NCA, MDP, IL20Rα,Brevican, EphB2R, ASLG659, PSCA, GEDA, BAFF-R, CD22, CD79a, CXCR5,HLA-DOB, P2X5, CD72, LY64, FcRH1, FcRH5, TENB2, PMEL17, TMEFF1,GDNF-Ra1, Ly6E, TMEM46, Ly6G6D, LGR5, RET, Ly6K, GPR19, GPR54, ASPHD1,Tyrosinase, TMEM118, GPR172A, CD33 and CLL-1. In yet other aspects, Abis a cysteine-engineered antibody. Suitable cysteine-engineered antibodyis a mutant selected from LC K149C, HC A140, HC A118C, and HC L177C. Instill other aspects, Ab is selected from anti-HER2 4D5, anti-CD22,anti-CD33, anti-Ly6E, anti-Napi3b, anti-HER2 7C2, and anti-CLL-1.

Drug loading is represented by p, the number of drug moieties perantibody in a molecule of Formula I. Drug loading may range from 1 to 20drug moieties (D) per antibody. Antibody-drug conjugates of Formula Iinclude collections of antibodies conjugated with a range of drugmoieties, from 1 to 20. In some embodiments, the number of drug moietiesthat can be conjugated to an antibody is limited by the number of freecysteine residues. In some embodiments, free cysteine residues areintroduced into the antibody amino acid sequence by the methodsdescribed herein. In such aspects, p may be 1, 2, 3, 4, 5, 6, 7, or 8,and ranges thereof, such as from 1 to 8 or from 2 to 5. In any suchaspect, p and n are equal (i.e., p=n=1, 2, 3, 4, 5, 6, 7, or 8, or somerange there between). Exemplary antibody-drug conjugates of Formula Iinclude, but are not limited to, antibodies that have 1, 2, 3, or 4engineered cysteine amino acids (Lyon, R. et al. (2012) Methods inEnzym. 502:123-138). In some embodiments, one or more free cysteineresidues are already present in an antibody, without the use ofengineering, in which case the existing free cysteine residues may beused to conjugate the antibody to a drug. In some embodiments, anantibody is exposed to reducing conditions prior to conjugation of theantibody in order to generate one or more free cysteine residues. Theaverage number of drug moieties per antibody (DAR) in preparations ofantibody-drug conjugates from conjugation reactions may be characterizedby conventional means such as mass spectroscopy, ELISA assay, and HPLC.The quantitative distribution of antibody-drug conjugates in terms of pmay also be determined. In some instances, separation, purification, andcharacterization of homogeneous antibody-drug conjugates where p is acertain value from antibody-drug conjugates with other drug loadings maybe achieved by means such as reverse phase HPLC or electrophoresis.

For some antibody-drug conjugates, p may be limited by the number ofattachment sites on the antibody. For example, where the attachment is acysteine thiol, as in certain exemplary embodiments described herein, anantibody may have only one or a limited number of cysteine thiol groups,or may have only one or a limited number of sufficiently reactive thiolgroups, to which the drug may be attached. In certain embodiments,higher drug loading, e.g. p>5, may cause aggregation, insolubility,toxicity, or loss of cellular permeability of certain antibody-drugconjugates. In certain embodiments, the average drug loading for anantibody-drug conjugate ranges from 1 to about 8; from about 2 to about6; or from about 3 to about 5. Indeed, it has been shown that forcertain antibody-drug conjugates, the optimal ratio of drug moieties perantibody may be less than 8, and may be between about 2 to about 5 (see,e.g., U.S. Pat. No. 7,498,298).

In certain embodiments, fewer than the theoretical maximum of drugmoieties are conjugated to an antibody during a conjugation reaction. Anantibody may contain, for example, lysine residues that do not reactwith the drug, as discussed herein. Generally, antibodies do not containmany free and reactive cysteine thiol groups which may be linked to adrug moiety; indeed most cysteine thiol residues in antibodies exist asdisulfide bridges. In certain embodiments, an antibody may be reducedwith a reducing agent such as dithiothreitol (DTT) ortricarbonylethylphosphine (TCEP), under partial or total reducingconditions, to generate reactive cysteine thiol groups. In certainembodiments, an antibody is subjected to denaturing conditions to revealreactive nucleophilic groups such as lysine or cysteine.

The loading (drug/antibody ratio) of an antibody-drug conjugate may becontrolled in different ways, and for example, by: (i) limiting themolar excess of the drug relative to antibody, (ii) limiting theconjugation reaction time or temperature, and (iii) partial or limitingreductive conditions for cysteine thiol modification.

It is to be understood that where more than one nucleophilic groupreacts with a drug, then the resulting product is a mixture ofantibody-drug conjugate compounds with a distribution of one or moredrug moieties attached to an antibody. The average number of drugs perantibody may be calculated from the mixture by a dual ELISA antibodyassay, which is specific for antibody and specific for the drug.Individual antibody-drug conjugate molecules may be identified in themixture by mass spectroscopy and separated by HPLC, e.g. hydrophobicinteraction chromatography (see, e.g., McDonagh et al. (2006) Prot.Engr. Design & Selection 19(7):299-307; Hamblen et al. (2004) Clin.Cancer Res. 10:7063-7070; Hamblen, K. J., et al. “Effect of drug loadingon the pharmacology, pharmacokinetics, and toxicity of an anti-CD30antibody-drug conjugate,” Abstract No. 624, American Association forCancer Research, 2004 Annual Meeting, Mar. 27-31, 2004, Proceedings ofthe AACR, Volume 45, March 2004; Alley, S. C., et al. “Controlling thelocation of drug attachment in antibody-drug conjugates,” Abstract No.627, American Association for Cancer Research, 2004 Annual Meeting, Mar.27-31, 2004, Proceedings of the AACR, Volume 45, March 2004). In certainembodiments, a homogeneous antibody-drug conjugate with a single loadingvalue may be isolated from the conjugation mixture by electrophoresis orchromatography.

In some other aspects of the present disclosure, calicheamicinderivative compositions comprising a thiopyridyl leaving group or abenzimidazole leaving group are provided. Such compositions are termed“activated calicheamicin.” Activated calicheamicin may then beconjugated with an antibody as described elsewhere herein. Exemplarycalicheamicin derivative-leaving group compositions are depicted belowas Formulae I and

In such aspects, R is selected from H, —C(O)R¹, —C(O)NR¹R², —S(O)₂R¹,and —S(O)₂NR²R¹; R¹ and R² are independently selected from C₁-C₆ alkyland C₆-C₂₀ aryl; R³ is selected from NO₂, Cl, F, CN, CO₂H, and Br; and qis 0, 1, or 2.

In an exemplary embodiment, R is —C(O)CH₃.

In an exemplary embodiment, R³ is NO₂ and q is 1.

In one exemplary embodiment, the drug intermediate has the Formula Ia:

In another exemplary embodiment, the drug intermediate has the formulaIIa:

Formation of activated calicheamicin is represented by the followingscheme where X is a leaving group as defined elsewhere herein:

In the reaction, the methyltrisulfide moiety of calicheamicin is reactedwith the leaving group thiol moiety to form a disulfide where S_(c)refers to a calicheamicin sulfur atom and S, refers to a leaving groupsulfur atom. Examples of this reaction are given, for instance, in U.S.Pat. Nos. 5,053,394, 5,712,374, 5,714,586, 5,739,116 and 5,767,285. Nonlimiting examples of suitable solvents for forming reaction mixturesinclude polar aprotic solvents such as acetonitrile, tetrahydrofuran,ethyl acetate, acetone, N,N-dimethylformamide, dimethylsulfoxide anddichloromethane. Calicheamicin concentration in the reaction mixture isnot narrowly critical and may suitably vary from about 0.0005 to about0.01 mmol/mL, such as about 0.0005, about 0.001, about 0.005, or about0.01 mmol/mL. The leaving group is present in stoichiometric excess,such as about 1.1:1 mole/mole, about 1.5:1 mole/mole, about 2:1mole/mole, about 2.5:1 mole/mole or about 3:1 mole/mole. The reactiontemperature is suitably about −30° C., about −20° C., about −10° C.,about 0° C., or about 10° C., and ranges thereof, such as from about−30° C. to about 10° C., from about −30° C. to about 0° C., or fromabout −30° C. to about −10° C. The reaction time to completion maysuitably vary from about 4 hours to about 4 days, such as from about 8hours to about 36 hours or from about 18 hours to about 36 hours.

In some aspects of the disclosure, activated calicheamicin may bepurified and isolated as a solid. Purification and isolation methods areknown in the art and include precipitation, crystallization, filtration,centrifugation, ultrafiltration, and various chromatographic techniques.Chromatography can involve any number of methods including, e.g.:reverse-phase and normal phase; size exclusion; ion exchange; high,medium and low pressure liquid chromatography methods and apparatus;small scale analytical; simulated moving bed (SMB) and preparative thinor thick layer chromatography, as well as techniques of small scale thinlayer and flash chromatography. In some such aspects, the completedreaction mixture may be evaporated to dryness followed by re-dissolutionin a polar aprotic solvent. The solution may be filtered and thenprecipitated by combining the solution with a nonpolar antisolvent suchas, for instance, hexane or cyclohexane. The precipitate may then becollected by filtration, optionally washed, and then dried.

Formation of calicheamicin-antibody conjugates is represented by thefollowing scheme:

In the scheme, p refers to the number of activated calicheamicinequivalents, S_(c) is a calicheamicin sulfur atom, X is a leaving groupas defined elsewhere herein, S_(x) is a leaving group sulfur atom, Ab isan antibody as described elsewhere herein, S_(a)H is an Ab freesulfhydryl moiety suitable for conjugation as described elsewhereherein, n is the number of equivalents of Ab free sulfhydryl moietiesper Ab equivalent. Each p and n are defined elsewhere herein. In someaspects, Ab-(S_(a)H)_(n) may be an cysteine engineered antibody asdescribed elsewhere herein and/or may be treated with a reducing agentfor reactivity in the conjugation reaction. The Ab is dissolved in aphysiological buffer system known in the art that will not adverselyimpact the stability or antigen-binding specificity of the antibody. Insome aspects, phosphate buffered saline is used. Activated calicheamicinis dissolved in a solvent system comprising at least one polar aproticsolvent as described elsewhere herein. In some such aspects, activatedcalicheamicin is dissolved to a concentration of about 5 mM, 10 mM,about 20 mM, about 30 mM, about 40 mM or about 50 mM, and ranges thereofsuch as from about 50 mM to about 50 mM or from about 10 mM to about 30mM in pH 8 Tris buffer (e.g., 50 mM Tris). In some aspects, activatedcalicheamicin is dissolved in DMSO or acetonitrile, or in DMSO. In theconjugation reaction, an equivalent excess of activated calicheamicinsolution is diluted and combined with chilled antibody solution (e.g.from about 1° C. to about 10° C.). The activated calicheamicin solutionmay suitably be diluted with at least one polar aprotic solvent and atleast one polar protic solvent, examples of which include water,methanol, ethanol, n-propanol, and acetic acid. In some particularaspects the activated calicheamicin is dissolved in DMSO and dilutedwith acetonitrile and water prior to admixture with the antibodysolution. The equivalents of calicheamicin to antibody may suitably beabout 1.5:1, about 3:1, about 5:1, about 10:1 about 15:1 or about 20:1,and ranges thereof, such as from about 1.5:1 to about 20:1 from about1.5:1 to about 15:1, from about 1.5:1 to about 10:1, from about 3:1 toabout 15:1, from about 3:1 to about 10:1, from about 5:1 to about 15:1or from about 5:1 to about 10:1. The reaction may suitably be monitoredfor completion by methods known in the art, such as LC-MS (as describedelsewhere herein), and the reaction is typically complete in from about1 hour to about 24 hours. After the reaction is complete, a reagent isadded to the reaction mixture to quench the reaction and cap unreactedantibody thiol groups. An example of a suitable reagent is maleimide.

Following conjugation, the antibody-calicheamicin conjugates may bepurified and separated from unconjugated reactants and/or conjugateaggregates by purification methods known in the art such as, for exampleand not limited to, size exclusion chromatography, hydrophobicinteraction chromatography, ion exchange chromatography,chromatofocusing, ultrafiltration, centrifugal ultrafiltration, andcombinations thereof. For instance, purification may be preceded bydiluting the antibody-calicheamicin conjugate, such in 20 mM sodiumsuccinate, pH 5. The diluted solution is applied to a cation exchangecolumn followed by washing with, e.g., at least 10 column volumes of 20mM sodium succinate, pH 5. The conjugate may be suitably eluted withPBS.

Pharmaceutical Formulations

Pharmaceutical formulations of therapeutic antibody-drug conjugates ofthe invention are typically prepared for parenteral administration, i.e.bolus, intravenous, intratumor injection in a unit dosage injectableform with the desired degree of purity and with one or more optionalpharmaceutically acceptable carriers, excipient, and/or vehicles(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)),in the form of lyophilized formulations or aqueous solutions.Pharmaceutically acceptable carriers are generally nontoxic torecipients at the dosages and concentrations employed, and include, butare not limited to: buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride; benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as polyethylene glycol(PEG). Exemplary pharmaceutically acceptable carriers herein furtherinclude insterstitial drug dispersion agents such as solubleneutral-active hyaluronidase glycoproteins (sHASEGP), for example, humansoluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®,Baxter International, Inc.). Certain exemplary sHASEGPs and methods ofuse, including rHuPH20, are described in US Patent Publication Nos.2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined withone or more additional glycosaminoglycanases, such as chondroitinases.

Exemplary lyophilized antibody or immunoconjugate formulations aredescribed in U.S. Pat. No. 6,267,958. Aqueous antibody orimmunoconjugate formulations include those described in U.S. Pat. No.6,171,586 and WO2006/044908, the latter formulations including ahistidine-acetate buffer.

The formulation herein may also contain more than one active ingredientas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody or immunoconjugate, whichmatrices are in the form of shaped articles, e.g. films, ormicrocapsules.

The formulations to be used for in vivo administration are generallysterile. Sterility may be readily accomplished, e.g., by filtrationthrough sterile filtration membranes.

Antibody-Drug Conjugate Methods of Treatment

It is contemplated that the antibody-drug conjugates of the presentinvention may be used to treat various diseases or disorders, e.g.characterized by the overexpression of a tumor antigen. Exemplaryconditions or hyperproliferative disorders include benign or malignantsolid tumors and hematological disorders such as leukemia and lymphoidmalignancies. Others include neuronal, glial, astrocytal, hypothalamic,glandular, macrophagal, epithelial, stromal, blastocoelic, inflammatory,angiogenic and immunologic, including autoimmune, disorders.

In one aspect, an antibody-drug conjugate provided herein is used in amethod of inhibiting proliferation of a cancer cell, the methodcomprising exposing the cell to the antibody-drug conjugate underconditions permissive for binding of the antibody or antibody-drugconjugates to a tumor-associated antigen on the surface of the cell,thereby inhibiting the proliferation of the cell. In certainembodiments, the method is an in vitro or an in vivo method. In furtherembodiments, the cell is a lymphocyte, lymphoblast, monocyte, ormyelomonocyte cell.

Inhibition of cell proliferation in vitro may be assayed using theCellTiter-Glo™ Luminescent Cell Viability Assay, which is commerciallyavailable from Promega (Madison, Wis.). That assay determines the numberof viable cells in culture based on quantitation of ATP present, whichis an indication of metabolically active cells. See Crouch et al. (1993)J. Immunol. Meth. 160:81-88, U.S. Pat. No. 6,602,677. The assay may beconducted in 96- or 384-well format, making it amenable to automatedhigh-throughput screening (HTS). See Cree et al. (1995) AntiCancer Drugs6:398-404. The assay procedure involves adding a single reagent(CellTiter-Glo® Reagent) directly to cultured cells. This results incell lysis and generation of a luminescent signal produced by aluciferase reaction. The luminescent signal is proportional to theamount of ATP present, which is directly proportional to the number ofviable cells present in culture. Data can be recorded by luminometer orCCD camera imaging device. The luminescence output is expressed asrelative light units (RLU).

In another aspect, an antibody-drug conjugate for use as a medicament isprovided. In further aspects, an antibody-drug conjugate for use in amethod of treatment is provided. In certain embodiments, anantibody-drug conjugate for use in treating cancer is provided. Incertain embodiments, the invention provides an antibody-drug conjugatefor use in a method of treating an individual comprising administeringto the individual an effective amount of the antibody-drug conjugate. Inone such embodiment, the method further comprises administering to theindividual an effective amount of at least one additional therapeuticagent, e.g., as described herein.

In a further aspect, the invention provides for the use of anantibody-drug conjugate in the manufacture or preparation of amedicament. In one embodiment, the medicament is for treatment ofCLL-1-positive cancer. In a further embodiment, the medicament is foruse in a method of treating CLL-1-positive cancer, the method comprisingadministering to an individual having CLL-1-positive cancer an effectiveamount of the medicament. In one such embodiment, the method furthercomprises administering to the individual an effective amount of atleast one additional therapeutic agent, e.g., as described herein.

In a further aspect, the invention provides a method for treatingcancer. In one embodiment, the method comprises administering to anindividual having such cancer, characterized by detection of atumor-associated expressing antigen, an effective amount of anantibody-drug conjugate of the invention. In one such embodiment, themethod further comprises administering to the individual an effectiveamount of at least one additional therapeutic agent, as describedherein.

Antibody-drug conjugates of the invention can be used either alone or incombination with other agents in a therapy. For instance, an antibody orimmunoconjugate of the invention may be co-administered with at leastone additional therapeutic agent. In some embodiments, the additionaltherapeutic agent is an anthracycline. In some embodiments, theanthracycline is daunorubicin or idarubicin. In some embodiments, theadditional therapeutic agent is cytarabine. In some embodiments, theadditional therapeutic agent is cladribine. In some embodiments, theadditional therapeutic agent is fludarabine or topotecan. In someembodiments, the additional therapeutic agent is 5-azacytidine ordecitabine.

Such combination therapies noted herein encompass combinedadministration (where two or more therapeutic agents are included in thesame or separate formulations), and separate administration, in whichcase, administration of the antibody or immunoconjugate of the inventioncan occur prior to, simultaneously, and/or following, administration ofthe additional therapeutic agent and/or adjuvant. Antibodies orimmunoconjugates of the invention can also be used in combination withradiation therapy.

An antibody or immunoconjugate of the invention (and any additionaltherapeutic agent) can be administered by any suitable means, includingparenteral, intrapulmonary, and intranasal, and, if desired for localtreatment, intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. Dosing can be by any suitable route, e.g.by injections, such as intravenous or subcutaneous injections, dependingin part on whether the administration is brief or chronic. Variousdosing schedules including but not limited to single or multipleadministrations over various time-points, bolus administration, andpulse infusion are contemplated herein.

Antibodies or immunoconjugates of the invention would be formulated,dosed, and administered in a fashion consistent with good medicalpractice. Factors for consideration in this context include theparticular disorder being treated, the particular mammal being treated,the clinical condition of the individual patient, the cause of thedisorder, the site of delivery of the agent, the method ofadministration, the scheduling of administration, and other factorsknown to medical practitioners. The antibody or immunoconjugate need notbe, but is optionally formulated with one or more agents currently usedto prevent or treat the disorder in question. The effective amount ofsuch other agents depends on the amount of antibody or immunoconjugatepresent in the formulation, the type of disorder or treatment, and otherfactors discussed herein. These are generally used in the same dosagesand with administration routes as described herein, or about from 1 to99% of the dosages described herein, or in any dosage and by any routethat is empirically/clinically determined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of anantibody or immunoconjugate of the invention (when used alone or incombination with one or more other additional therapeutic agents) willdepend on the type of disease to be treated, the type of antibody orimmunoconjugate, the severity and course of the disease, whether theantibody or immunoconjugate is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the antibody or immunoconjugate, and the discretion ofthe attending physician. The antibody or immunoconjugate is suitablyadministered to the patient at one time or over a series of treatments.Depending on the type and severity of the disease, about 1 μg/kg to 15mg/kg (e.g. 0.1 mg/kg-10 mg/kg) of antibody or immunoconjugate can be aninitial candidate dosage for administration to the patient, whether, forexample, by one or more separate administrations, or by continuousinfusion. One typical daily dosage might range from about 1 μg/kg to 100mg/kg or more, depending on the factors mentioned herein. For repeatedadministrations over several days or longer, depending on the condition,the treatment would generally be sustained until a desired suppressionof disease symptoms occurs. One exemplary dosage of the antibody orimmunoconjugate would be in the range from about 0.05 mg/kg to about 10mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kgor 10 mg/kg (or any combination thereof) may be administered to thepatient. Such doses may be administered intermittently, e.g. every weekor every three weeks (e.g. such that the patient receives from about twoto about twenty, or e.g. about six doses of the antibody). An initialhigher loading dose, followed by one or more lower doses may beadministered. However, other dosage regimens may be useful. The progressof this therapy is easily monitored by conventional techniques andassays.

Intracellular release of calicheamicin from the antibody-calicheamicinconjugate in a target cell is believed to result from reductive cleavageof the disulfide bond by glutathione. Glutathione-mediated releaseprovides for advantages as compared to certain linkers known in theprior art, such as acid-labile hydrazine linkers. More particularly,blood concentration of glutathione is known to be very low, such as inthe micromolar range, whereas intracellular glutathione concentration istypically up to three orders of magnitude greater, such as in themillimolar range. It is further believed that glutathione concentrationin cancer cells is even greater due to increased activity of reductiveenzymes. Therefore, it is believed that the calicheamicin-antibodyconjugates of the present disclosure provide for improved stability inthe bloodstream and for improved intracellular release rates.

Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described herein is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, IV solution bags, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition which is by itself or combined with anothercomposition effective for treating, preventing and/or diagnosing thedisorder and may have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is an antibody or immunoconjugate of the invention. Thelabel or package insert indicates that the composition is used fortreating the condition of choice. Moreover, the article of manufacturemay comprise (a) a first container with a composition contained therein,wherein the composition comprises an antibody or immunoconjugate of theinvention; and (b) a second container with a composition containedtherein, wherein the composition comprises a further cytotoxic orotherwise therapeutic agent. The article of manufacture in thisembodiment of the invention may further comprise a package insertindicating that the compositions can be used to treat a particularcondition. Alternatively, or additionally, the article of manufacturemay further comprise a second (or third) container comprising apharmaceutically-acceptable buffer, such as bacteriostatic water forinjection (BWFI), phosphate-buffered saline, Ringer's solution ordextrose solution. It may further include other materials desirable froma commercial and user standpoint, including other buffers, diluents,filters, needles, and syringes.

Examples

The following are examples of methods and compositions of the invention.It is understood that various other embodiments may be practiced, giventhe general description provided herein.

Example 1—Preparation of Cysteine Engineered Antibodies

For large scale antibody production, antibodies were produced in CHOcells. Vectors coding for VL and VH were transfected into CHO cells andIgG was purified from cell culture media by protein A affinitychromatography.

As initially isolated, the engineered cysteine residues in antibodiesexist as mixed disulfides with cellular thiols (e.g., glutathione) andare thus unavailable for conjugation. Partial reduction of theseantibodies (e.g., with DTT), purification, and reoxidation withdehydroascorbic acid (DHAA) gives antibodies with free cysteinesulfhydryl groups available for conjugation, as previously described,e.g., in Junutula et al. (2008) Nat. Biotechnol. 26:925-932 and US2011/0301334. Briefly, the antibodies were combined with the activatedcalicheamicin drug moiety to allow conjugation to the free cysteineresidues of the antibody. After several hours, the antibody-drugconjugates were purified.

Under certain conditions, the cysteine engineered antibodies were madereactive for conjugation with drugs by treatment with a reducing agentsuch as DTT (Cleland's reagent, dithiothreitol) or TCEP(tris(2-carboxyethyl)phosphine hydrochloride; Getz et al. (1999) Anal.Biochem. Vol 273:73-80; Soltec Ventures, Beverly, Mass.) in 50 mM TrispH 7.5 with 2 mM EDTA for 3 hrs at 37° C. or overnight at roomtemperature. Full length, cysteine engineered monoclonal antibodies(THIOMAB™) expressed in CHO cells (Gomez et al. (2010) Biotechnology andBioeng. 105(4):748-760; Gomez et al. (2010) Biotechnol. Prog.26:1438-1445) were reduced, for example with about a 50 fold excess ofDTT overnight at room temperature to reduce disulfide bonds which mayform between the newly introduced cysteine residues and the cysteinepresent in the culture media. The reduced THIOMAB™ was diluted andloaded onto a HiTrap S column in 10 mM sodium acetate, pH 5, and elutedwith PBS containing 0.3M sodium chloride. Alternatively, the antibodywas acidified by addition of 1/20^(th) volume of 10% acetic acid,diluted with 10 mM succinate pH 5, loaded onto the column and thenwashed with 10 column volumes of succinate buffer. The column was elutedwith 50 mM Tris pH7.5, 2 mM EDTA.

Light chain amino acids are numbered according to Kabat (Kabat et al.,Sequences of proteins of immunological interest, (1991) 5th Ed., US Deptof Health and Human Service, National Institutes of Health, Bethesda,Md.). Heavy chain amino acids are numbered according to the EU numberingsystem (Edelman et al. (1969) Proc. Natl. Acad. of Sci. 63(1):78-85),except where noted as the Kabat system. Single letter amino acidabbreviations are used.

Full length, cysteine engineered monoclonal antibodies (THIOMAB™)expressed in CHO cells bear cysteine adducts (cystines) orglutathionylated on the engineered cysteines due to cell cultureconditions. To liberate the reactive thiol groups of the engineeredcysteines, the THIOMAB™ was dissolved in 500 mM sodium borate and 500 mMsodium chloride at about pH 8.0 and reduced with about a 50-100 foldexcess of 1 mM TCEP (tris(2-carboxyethyl)phosphine hydrochloride (Getzet al. (1999) Anal. Biochem. Vol 273:73-80; Soltec Ventures, Beverly,Mass.) for about 1-2 hrs at 37° C. Alternatively, DTT was used asreducing agent. The formation of inter-chain disulfide bonds wasmonitored either by non-reducing SDS-PAGE or by denaturing reverse phaseHPLC PLRP column chromatography. The reduced THIOMAB™ was diluted andloaded onto a HiTrap SP FF column in 10 mM sodium acetate, pH 5, andeluted with PBS containing 0.3M sodium chloride, or 50 mM Tris-Cl, pH7.5 containing 150 mM sodium chloride.

Disulfide bonds were reestablished between cysteine residues present inthe parent Mab by carrying out reoxidation. The eluted reduced THIOMAB™was treated with 15× or 2 mM dehydroascorbic acid (dhAA) at pH 7 forabout 3 hours or for about 3 hrs in 50 mM Tris-Cl, pH 7.5, or with 200nM to 2 mM aqueous copper sulfate (CuSO₄) at room temperature overnight.Other oxidants, i.e. oxidizing agents, and oxidizing conditions, whichare known in the art may be used. Ambient air oxidation may also beeffective. This mild, partial reoxidation step formed intrachaindisulfides efficiently with high fidelity. The buffer was exchanged byelution over Sephadex G25 resin and eluted with PBS with 1 mM DTPA. Thethiol/antibody value was checked by determining the reduced antibodyconcentration from the absorbance at 280 nm of the solution and thethiol concentration by reaction with DTNB (Aldrich, Milwaukee, Wis.) anddetermination of the absorbance at 412 nm.

Liquid chromatography/Mass Spectrometric Analysis was performed on a TSQQuantum Triple Quadrupole™ mass spectrometer with extended mass range(Thermo Electron, San Jose Calif.). Samples were chromatographed on aPRLP-S®, 1000 A, microbore column (50 mm×2.1 mm, Polymer Laboratories,Shropshire, UK) heated to 75° C. A linear gradient from 30-40% B(solvent A: 0.05% TFA in water, solvent B: 0.04% TFA in acetonitrile)was used and the eluent was directly ionized using the electrospraysource. Data was collected by the Xcalibur® data system anddeconvolution was performed using ProMass® (Novatia, LLC, New Jersey).Prior to LC/MS analysis, antibodies or drug conjugates (50 micrograms)were treated with PNGase F (2 units/ml; PROzyme, San Leandro, Calif.)for 2 hours at 37° C. to remove N-linked carbohydrates.

Hydrophobic Interaction Chromatography (HIC) samples were injected ontoa Butyl HIC NPR column (2.5 micron particle size, 4.6 mm×3.5 cm) (TosohBioscience) and eluted with a linear gradient from 0 to 70% B at 0.8ml/min (A: 1.5 M ammonium sulfate in 50 mM potassium phosphate, pH 7, B:50 mM potassium phosphate pH 7, 20% isopropanol). An Agilent 1100 seriesHPLC system equipped with a multi wavelength detector and Chemstationsoftware was used to resolve and quantitate antibody species withdifferent ratios of drugs per antibody.

Example 2—Conjugation of Calicheamicin to Antibodies

After the reduction and reoxidation procedures of Example 1, thecysteine-engineered antibody (THIOMAB™) is dissolved in PBS (phosphatebuffered saline) buffer and chilled on ice. An excess, from about 1.5molar to 20 equivalents of a calicheamicin, activated with athiol-reactive pyridyl disulfide group, is dissolved in DMSO, diluted inacetonitrile and water, and added to the chilled reduced, reoxidizedantibody in PBS. Typically the drug is added from a DMSO stock at aconcentration of about 20 mM in 50 mM Tris, pH 8, to the antibody andmonitored until the reaction is complete from about 1 to about 24 hoursas determined by LC-MS analysis of the reaction mixture. When thereaction is complete, an excess of maleimide is added to quench thereaction and cap any unreacted antibody thiol groups. The conjugationmixture may be loaded and eluted through a HiTrap SP FF column to removeexcess drug and other impurities. The reaction mixture is concentratedby centrifugal ultrafiltration and the cysteine engineered antibody-drugconjugate is purified and desalted by elution through G25 resin in PBS,filtered through 0.2 μm filters under sterile conditions, and frozen forstorage.

For example, the crude antibody-drug conjugate is applied to a cationexchange column after dilution with 20 mM sodium succinate, pH 5. Thecolumn was washed with at least 10 column volumes of 20 mM sodiumsuccinate, pH 5, and the antibody was eluted with PBS. The antibody-drugconjugates were formulated into 20 mM His/acetate, pH 5, with 240 mMsucrose using gel filtration columns. The antibody-drug conjugates werecharacterized by UV spectroscopy to determine protein concentration,analytical SEC (size-exclusion chromatography) for aggregation analysisand LC-MS before and after treatment with Lysine C endopeptidase.

Size exclusion chromatography is performed using a Shodex KW802.5 columnin 0.2M potassium phosphate pH 6.2 with 0.25 mM potassium chloride and15% IPA at a flow rate of 0.75 ml/min. Aggregation state of theconjugate was determined by integration of eluted peak area absorbanceat 280 nm.

LC-MS analysis may be performed using an Agilent QTOF 6520 ESIinstrument. As an example, the antibody-drug conjugate is treated with1:500 w/w Endoproteinase Lys C (Promega) in Tris, pH 7.5, for 30 min at37° C. The resulting cleavage fragments are loaded onto a 1000 Å(Angstrom), 8 μm (micron) PLRP-S (highly cross-linked polystyrene)column heated to 80° C. and eluted with a gradient of 30% B to 40% B in5 minutes. Mobile phase A was H₂O with 0.05% TFA and mobile phase B wasacetonitrile with 0.04% TFA. The flow rate was 0.5 ml/min. Proteinelution was monitored by UV absorbance detection at 280 nm prior toelectrospray ionization and MS analysis. Chromatographic resolution ofthe unconjugated Fc fragment, residual unconjugated Fab and drugged Fabwas usually achieved. The obtained m/z spectra were deconvoluted usingMass Hunter™ software (Agilent Technologies) to calculate the mass ofthe antibody fragments.

Example 3—In Vitro Cell Proliferation Assay

Efficacy of the antibody-drug conjugates Thio Hu Anti-CD22 10F4v3 LCK149C calicheamicin and Thio Hu Anti-Ly6E 9B12.v12 LC K149Ccalicheamicin was measured by a cell proliferation assay employing thefollowing protocol (CELLTITER GLO™ Luminescent Cell Viability Assay,Promega Corp. Technical Bulletin TB288; Mendoza et al. (2002) CancerRes. 62:5485-5488):

1. An aliquot of 100 μl of cell culture containing about 10⁴ cells(CD22-positive BJAB, CD22-positive WSU-DLCL2 or Jurkat) in medium wasdeposited in each well of a 96-well, opaque-walled plate.

2. Control wells were prepared containing medium and without cells.

3. Antibody-drug conjugate was added to the experimental wells andincubated for 3-5 days.

4. The plates were equilibrated to room temperature for approximately 30minutes.

5. A volume of CELLTITER GLO™ Reagent equal to the volume of cellculture medium present in each well was added.

6. The contents were mixed for 2 minutes on an orbital shaker to inducecell lysis.

7. The plate was incubated at room temperature for 10 minutes tostabilize the luminescence signal.

8. Luminescence was recorded and reported in graphs as RLU=relativeluminescence units.

Data was plotted and illustrated in FIGS. 3A to 3C as the mean ofluminescence for each set of replicates, with standard deviation errorbars. The protocol is a modification of the CELLTITER GLO™ LuminescentCell.

Example 4—Tumor Growth Inhibition, In Vivo Efficacy in High ExpressingHER2 Transgenic Explant Mice

Tumors were established and allowed to grow to 150-200 mm³ in volume (asmeasured using calipers) before a single treatment on day 0. Tumorvolume was measured using calipers according to the formula: V(mm³)=0.5A×B², where A and B are the long and short diameters,respectively. Mice were euthanized before tumor volume reached 3000 mm³or when tumors showed signs of impending ulceration. Data collected fromeach experimental group (10 mice per group) was expressed as mean±SE.

The Fo5 mouse mammary tumor model was employed to evaluate the in vivoefficacy of antibody-drug conjugates of the invention after single doseintravenous injections, and as described previously (Phillips GDL, Li GM, Dugger D L, et al. Targeting HER2-Positive Breast Cancer withTrastuzumab-DM1, an Antibody-Cytotoxic Drug Conjugate. (2008) CancerRes. 68:9280-90), incorporated by reference herein. Anti-Her2antibody-drug conjugates were tested with the Fo5 model, a transgenicmouse model in which the human HER2 gene is over-expressed in mammaryepithelium under transcriptional regulation of the murine mammary tumorvirus promoter (MMTV-HER2). The HER2 over-expression causes spontaneousdevelopment of a mammary tumor. The mammary tumor of one of thesefounder animals (founder #5 [Fo5]) was propagated in subsequentgenerations of FVB mice by serial transplantation of tumor fragments(˜2×2 mm in size). All studies were conducted in accordance with theGuide for the Care and Use of Laboratory Animals. Each antibody-drugconjugate (single dose) was dosed in nine animals intravenously at thestart of the study, and 14 days post-transplant. Initial tumor size wasabout 200 mm³ volume.

Another mammary fat pad transplant efficacy model may be employed asdescribed (Chen et al. (2007) Cancer Res 67:4924-4932), evaluating tumorvolume after a single intravenous dose and using tumors excised from amouse bearing an intraperitoneal tumor, then serially passaged into themammary fat pads of recipient mice.

Cell lines that could be tested in this way include AU565, HCC1954,HCC1008, HCC2157, HCC202, HCC1419, HCC2218 and HCC1569.

Example 5—Efficacy of Thio Hu Anti-Ly6E LCK149C-p-Nitro-PDS-Calicheamicin Antibody-Drug Conjugates

Breast cancer cell line HCC1569 (CRL-2330) was obtained from AmericanType Culture Collection (ATCC, Manassas, Va.). The HCC1569×2 cell lineis a derivative of the parental HCC1569 cell line (ATCC, CRL-2330)optimized for growth in vivo. Parental HCC1569 cells were injectedsubcutaneously in the right flank of female NCR nude mice, one tumor washarvested, minced and grown in vitro resulting in a HCC1569 XI cellline. The HCC1569 XI line was injected again subcutaneously in the rightflank of female NCR nude mice in an effort to improve the growth of thecell line. A tumor from this study was collected and again adapted forin vitro growth to generate the HCC1569×2 cell line. This cell line andtumors derived from this line express Ly6E.

SCID Beige mice were inoculated in the right ⅔ mammary fat pad with 5million cells suspended in Hank's Balanced Salt Solution (HBSS) andmatrigel. When tumor volumes reached approximately 163-282 mm³ (day 0),the animals were randomized into groups of 5 mice each, and administereda single intravenous (IV) injection of either vehicle control or theantibody-drug conjugates at the following doses: 0.3 mg/kg, 1 mg/kg, 3mg/kg, 6 mg/kg and 10 mg/kg. One group of animals was administered 3mg/kg of Thio Hu anti-CD22 LC K149C-p-nitro-PDS-Calicheamicin. Tumorvolumes were measured twice per week until study end at 21 days. Tumorvolume was measured and calculated based on two dimensions, measuredusing calipers, and was expressed in mm³ according to the formula:V=0.5a×b², wherein a and b are the long and the short diameters of thetumor, respectively. To analyze the repeated measurement of tumorvolumes from the same animals over time, a mixed modeling approach wasused (see, e.g., Pinheiro J, et al. nlme: linear and nonlinear mixedeffects models. 2009; R package, version 3.1-96). This approach canaddress both repeated measurements and modest dropout rates due tonon-treatment related removal of animals before the study end. Cubicregression splines were used to fit a non-linear profile to the timecourses of log 2 tumor volume at each dose level. These non-linearprofiles were then related to dose within the mixed mode. All animalprotocols were approved by an Institutional Animal Care and UseCommittee (IACUC).

The experimental results are depicted in FIG. 1 and indicate that dosesof 1, 3, 6 and 10 mg/kg of Thio Hu anti-Ly6E LCK149C-p-nitro-PDS-Calicheamicin antibody-drug conjugates reduced tumorvolume over the course of the study.

Example 6—Efficacy of Thio Hu Anti-CD22 10F4v3 LCK149C-p-Nitro-PDS-Calicheamicin

The antitumor efficacy effect of Thio Hu anti-CD22 10F4v3 LCK149C-p-nitro-PDS-Calicheamicin conjugates in a mouse xenograft model ofWSU-DLCL2 tumors (diffuse large B-cell lymphoma cell line) was examined.

Female CB17 Fox Chase SCID mice were each inoculated in the right flankwith 20 million WSU-DLCL2 cells (DSMZ, German Collection ofMicroorganisms and Cell Cultures, Braunschweig, Germany) suspended inHank's Balanced Salt Solution (HBSS). When the xenograft tumors reachedan average tumor volume of 175-237 mm³ (day 0), the animals wererandomized into groups of 5 mice each and administered a singleintravenous (IV) injection of either vehicle control or theantibody-drug conjugate at the following doses: 0.3 mg/kg, 1 mg/kg, 3mg/kg, 6 mg/kg and 10 mg/kg. One group of animals was administered 3mg/kg of Thio Hu anti-LY6E 9B12.v12 LC K149-p-nitro-PDS-Calicheamicin.Tumor volumes were measured twice per week, as described elsewhereherein, until study end at 21 days. All animal protocols were approvedby an Institutional Animal Care and Use Committee (IACUC).

The experimental results are depicted in FIG. 2 and indicate that dosesof 1, 3, 6 and 10 mg/kg of Thio Hu anti-CD22 10F4v3 LCK149C-p-nitro-PDS-Calicheamicin reduced volume over the course of thestudy.

Example 7—Additional Efficacy Studies

Example 7 evaluated the efficacy of targeted control with Thio HuAnti-CD22 10F4v3 LC K149C calicheamicin conjugate against CD22 positiveBurkitt's lymphoma cells (“BJAB”) and against CD22 positive humandiffuse large B-cell lymphoma-derived cell line (WSU-DLCL2) versusnon-targeted control with Thio Hu Anti-Ly6E 9B12.v12 LC K149Ccalicheamicin conjugate. Each conjugate had an average drug to antibodyratio (“DAR”) of 1.7. The efficacy of non-targeted control for eachconjugate was also evaluated against Jurkat.

The IC₅₀ efficacy results for the BJAB, WSU-DLCL2 and Jurkat cells aredepicted in FIGS. 3A to 3C, respectively. The results show thattreatment of the CD22-positive BJAB and WSU-DLCL2 cell lines with ThioHu Anti-CD22 10F4v3 LC K149C-Calicheamicin provided double-digit potencythat is >1500-fold greater and >2000-fold greater than non-targetedcontrol with Thio Hu Anti-Ly6E 9B12.v12 LC K149C-Calicheamicin on BJABand WSU-DLCL2, respectively. The results further show that Thio HuAnti-CD22 10F4v3 LC K149C-Calicheamicin and Thio Hu Anti-Ly6E 9B12.v12LC K149C-Calicheamicin were each essentially non-efficacious on Jurkatcells. The IC₅₀ results are reported in the Table below where “ADC”refers to antibody-drug conjugate, “Thio Hu Anti-CD22” refers to Thio HuAnti-CD22 10F4v3 LC K149C calicheamicin, and “Thio Hu Anti-Ly6E” refersto Thio Hu Anti-Ly6E 9B12.v12 LC K149C calicheamicin.

WSU-DLDL2 BJAB IC₅₀ IC₅₀ Jurkat IC₅₀ ADC nM Ng/mL nM Ng/mL nM Ng/mL ThioHu Anti- 0.07 10.8 0.03 4.8 121.6 18229.5 CD22 Thio Hu Anti- 125.218777.4 82.7 12395.0 121.0 18138.9 Ly6E

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

When introducing elements of the present disclosure or the preferredembodiments(s) thereof, the articles “a”, “an”, “the” and “said” areintended to mean that there are one or more of the elements. The terms“comprising”, “including” and “having” are intended to be inclusive andmean that there may be additional elements other than the listedelements.

This written description uses examples to disclose the invention,including the best mode, and also to enable any person skilled in theart to practice the invention, including making and using any devices orsystems and performing any incorporated methods. The patentable scope ofthe invention is defined by the claims, and may include other examplesthat occur to those skilled in the art. Such other examples are intendedto be within the scope of the claims if they have structural elementsthat do not differ from the literal language of the claims, or if theyinclude equivalent structural elements with insubstantial differencesfrom the literal language of the claims.

What is claimed is:
 1. A drug intermediate composition of Formula I orFormula II:

wherein R is selected from H, —C(O)R¹, —C(O)NR¹R², —S(O)₂R¹, and—S(O)2NR²R¹; R¹ and R² are independently selected from C₁-C₆ alkyl andC₆-C₂₀ aryl; R³ is selected from NO₂, Cl, F, CN, CO₂H, and Br; and q is0, 1, or
 2. 2. The drug intermediate composition of claim 1 wherein R is—C(O)CH₃.
 3. The drug intermediate composition of claim 1 wherein R³ isNO₂ and q is
 1. 4. The drug intermediate composition of claim 3 havingFormula Ia:


5. The drug intermediate composition of claim 1 having Formula IIa:


6. An antibody-drug conjugate compound having Formula III:

or a pharmaceutically acceptable salt thereof, wherein R is selectedfrom H, —C(O)R¹, —C(O)NR¹R², —S(O)₂R¹, and —S(O)₂NR²R¹; R¹ and R² areindependently selected from C₁-C₆ alkyl and C₆-C₂₀ aryl; p is an integerfrom 1 to 8; and Ab is an antibody which binds to one or moretumor-associated antigens or cell-surface receptors selected from(1)-(53): (1) BMPR1B (bone morphogenetic protein receptor-type IB); (2)E16 (LAT1, SLC7A5); (3) STEAP1 (six transmembrane epithelial antigen ofprostate); (4) MUC16 (0772P, CA125); (5) MPF (MPF, MSLN, SMR,megakaryocyte potentiating factor, mesothelin); (6) Napi2b (NAPI-3B,NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate), member 2,type II sodium-dependent phosphate transporter 3b); (7) Sema 5b(F1110372, KIAA1445, Mm.42015, SEMASB, SEMAG, Semaphorin 5b Hlog, semadomain, seven thrombospondin repeats (type 1 and type 1-like),transmembrane domain (TM) and short cytoplasmic domain, (semaphorin)5B); (8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12,RIKEN cDNA 2700050C12 gene); (9) ETBR (Endothelin type B receptor); (10)MSG783 (RNF124, hypothetical protein F1120315); (11) STEAP2 (HGNC_8639,IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer associated gene1, prostate cancer associated protein 1, six transmembrane epithelialantigen of prostate 2, six transmembrane prostate protein); (12) TrpM4(BR22450, F1120041, TRPM4, TRPM4B, transient receptor potential cationchannel, subfamily M, member 4); (13) CRIPTO (CR, CR1, CRGF, CRIPTO,TDGF1, teratocarcinoma-derived growth factor); (14) CD21 (CR2(Complement receptor 2) or C3DR (C3d/Epstein Barr virus receptor) or Hs73792); (15) CD79b (CD79B, CD79β, IGb (immunoglobulin-associated beta),B29); (16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containingphosphatase anchor protein 1a), SPAP1B, SPAP1C); (17) HER2; (18) NCA;(19) MDP; (20) IL20Rα; (21) Brevican; (22) EphB2R; (23) ASLG659; (24)PSCA; (25) GEDA; (26) BAFF-R (B cell-activating factor receptor, BLySreceptor 3, BR3); (27) CD22 (B-cell receptor CD22-B isoform); (28) CD79a(CD79A, CD79α, immunoglobulin-associated alpha); (29) CXCR5 (Burkitt'slymphoma receptor 1); (30) HLA-DOB (Beta subunit of MHC class IImolecule (Ia antigen)); (31) P2X5 (Purinergic receptor P2X ligand-gatedion channel 5); (32) CD72 (B-cell differentiation antigen CD72, Lyb-2);(33) LY64 (Lymphocyte antigen 64 (RP105), type I membrane protein of theleucine rich repeat (LRR) family); (34) FcRH1 (Fc receptor-like protein1); (35) FcRH5 (IRTA2, Immunoglobulin superfamily receptor translocationassociated 2); (36) TENB2 (putative transmembrane proteoglycan); (37)PMEL17 (silver homolog; SILV; D12S53E; PMEL17; SI; SIL); (38) TMEFF1(transmembrane protein with EGF-like and two follistatin-like domains 1;Tomoregulin-1); (39) GDNF-Ra1 (GDNF family receptor alpha 1, GFRA1;GDNFR; GDNFRA; RETL1; TRNR1; RET1L; GDNFR-alpha1; GFR-ALPHA-1); (40)Ly6E (lymphocyte antigen 6 complex, locus E; Ly67, RIG-E, SCA-2, TSA-1);(41) TMEM46 (shisa homolog 2 (Xenopus laevis); SHISA2); (42) Ly6G6D(lymphocyte antigen 6 complex, locus G6D; Ly6-D, MEGT1); (43) LGR5(leucine-rich repeat-containing G protein-coupled receptor 5; GPR49,GPR67); (44) RET (ret proto-oncogene; MEN2A; HSCR1; MEN2B; MTC1; PTC;CDHF12; Hs.168114; RET51; RET-ELE1); (45) LY6K (lymphocyte antigen 6complex, locus K; LY6K; HSJ001348; FLJ35226); (46) GPR19 (Gprotein-coupled receptor 19; Mm.4787); (47) GPR54 (KISS1 receptor;KISS1R; GPR54; HOT7T175; AXOR12); (48) ASPHD1 (aspartatebeta-hydroxylase domain containing 1; LOC253982); (49) Tyrosinase (TYR;OCAIA; OCA1A; tyrosinase; SHEP3); (50) TMEM118 (ring finger protein,transmembrane 2; RNFT2; FLJ14627); (51) GPR172A (G protein-coupledreceptor 172k, GPCR41; FLJ11856; D15Ertd747e); (52) CD33; and (53)CLL-1.
 7. The antibody-drug conjugate compound according to claim 6,wherein Ab is a cysteine-engineered antibody.
 8. The antibody-drugconjugate compound according to claim 7, wherein the cysteine-engineeredantibody is a mutant selected from LC K149C, HC A140, HC A118C, and HCL177C.
 9. The antibody-drug conjugate compound according to claim 6,wherein Ab is selected from anti-HER2 4D5, anti-CD22, anti-CD33,anti-Ly6E, anti-Napi3b, anti-HER2 7C2, and anti-CLL-1.
 10. Theantibody-drug conjugate compound according to claim 6, wherein p is 1,2, 3, or
 4. 11. The antibody-drug conjugate compound according to claim6, comprising a mixture of the antibody-drug conjugate compounds,wherein the average drug loading per antibody in the mixture ofantibody-drug conjugate compounds is about 2 to about
 5. 12. Apharmaceutical composition comprising the antibody-drug conjugatecompound according to claim 6 and a pharmaceutically acceptable diluent,carrier or excipient.
 13. The pharmaceutical composition of claim 12,further comprising a therapeutically effective amount of achemotherapeutic agent.
 14. Use of an antibody-drug conjugate compoundaccording to claim 6 in the manufacture of a medicament for thetreatment of cancer in a mammal.
 15. A method of treating cancercomprising administering to a patient the pharmaceutical composition ofclaim
 14. 16. The method of claim 15 wherein the patient is administereda chemotherapeutic agent, in combination with the antibody-drugconjugate.
 17. An antibody-drug conjugate compound according to claim 6for use in a method for treating cancer.
 18. A method of making anantibody-drug conjugate compound of claim 6, the method comprising (a)reacting an antibody which binds to one or more tumor-associatedantigens or cell-surface receptors selected from (1)-(53): (1) BMPR1B(bone morphogenetic protein receptor-type IB); (2) E16 (LAT1, SLC7A5);(3) STEAP1 (six transmembrane epithelial antigen of prostate); (4) MUC16(0772P, CA125); (5) MPF (MPF, MSLN, SMR, megakaryocyte potentiatingfactor, mesothelin); (6) Napi2b (NAPI-3B, NPTIIb, SLC34A2, solutecarrier family 34 (sodium phosphate), member 2, type II sodium-dependentphosphate transporter 3b); (7) Sema 5b (F1110372, KIAA1445, Mm.42015,SEMASB, SEMAG, Semaphorin 5b Hlog, sema domain, seven thrombospondinrepeats (type 1 and type 1-like), transmembrane domain (TM) and shortcytoplasmic domain, (semaphorin) 5B); (8) PSCA hlg (2700050C12Rik,C530008O16Rik, RIKEN cDNA 2700050C12, RIKEN cDNA 2700050C12 gene); (9)ETBR (Endothelin type B receptor); (10) MSG783 (RNF124, hypotheticalprotein F1120315); (11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1,STEAP2, STMP, prostate cancer associated gene 1, prostate cancerassociated protein 1, six transmembrane epithelial antigen of prostate2, six transmembrane prostate protein); (12) TrpM4 (BR22450, F1120041,TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M,member 4); (13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1,teratocarcinoma-derived growth factor); (14) CD21 (CR2 (Complementreceptor 2) or C3DR (C3d/Epstein Barr virus receptor) or Hs 73792); (15)CD79b (CD79B, CD79β, IGb (immunoglobulin-associated beta), B29); (16)FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphatase anchorprotein 1a), SPAP1B, SPAP1C); (17) HER2; (18) NCA; (19) MDP; (20)IL20Rα; (21) Brevican; (22) EphB2R; (23) ASLG659; (24) PSCA; (25) GEDA;(26) BAFF-R (B cell-activating factor receptor, BLyS receptor 3, BR3);(27) CD22 (B-cell receptor CD22-B isoform); (28) CD79a (CD79A, CD79a,immunoglobulin-associated alpha); (29) CXCR5 (Burkitt's lymphomareceptor 1); (30) HLA-DOB (Beta subunit of MHC class II molecule (Iaantigen)); (31) P2X5 (Purinergic receptor P2X ligand-gated ion channel5); (32) CD72 (B-cell differentiation antigen CD72, Lyb-2); (33) LY64(Lymphocyte antigen 64 (RP105), type I membrane protein of the leucinerich repeat (LRR) family); (34) FcRH1 (Fc receptor-like protein 1); (35)FcRH5 (IRTA2, Immunoglobulin superfamily receptor translocationassociated 2); (36) TENB2 (putative transmembrane proteoglycan); (37)PMEL17 (silver homolog; SILV; D12S53E; PMEL17; SI; SIL); (38) TMEFF1(transmembrane protein with EGF-like and two follistatin-like domains I;Tomoregulin-1); (39) GDNF-Ra1 (GDNF family receptor alpha 1; GFRA1;GDNFR; GDNFRA; RETL1; TRNR1; RET1L; GDNFR-alpha1; GFR-ALPHA-1); (40)Ly6E (lymphocyte antigen 6 complex, locus E; Ly67, RIG-E, SCA-2, TSA-1);(41) TMEM46 (shisa homolog 2 (Xenopus laevis); SHISA2); (42) Ly6G6D(lymphocyte antigen 6 complex, locus G6D; Ly6-D, MEGT1); (43) LGR5(leucine-rich repeat-containing G protein-coupled receptor 5; GPR49,GPR67); (44) RET (ret proto-oncogene; MEN2A; HSCR1; MEN2B; MTC1; PTC;CDHF12; Hs.168114; RET51; RET-ELE1); (45) LY6K (lymphocyte antigen 6complex, locus K; LY6K; HSJ001348; FLJ35226); (46) GPR19 (Gprotein-coupled receptor 19; Mm.4787); (47) GPR54 (KISS1 receptor;KISS1R; GPR54; HOT7T175; AXOR12); (48) ASPHD1 (aspartatebeta-hydroxylase domain containing 1; LOC253982); (49) Tyrosinase (TYR;OCAIA; OCA1A; tyrosinase; SHEP3); (50) TMEM118 (ring finger protein,transmembrane 2; RNFT2; FLJ14627); (51) GPR172A (G protein-coupledreceptor 172A; GPCR41; FLJ11856; D15Ertd747e); (52) CD33; and (53) CLL-1(b) with a drug intermediate of Formula I or Formula II

wherein: R is selected from H, —C(O)R¹, —C(O)NR¹R², —S(O)₂R¹, and—S(O)2NR²R¹; R¹ and R² are independently selected from C₁-C₆ alkyl andC₆-C₂₀ aryl; R³ is selected from NO₂, Cl, F, CN, CO₂H, and Br; and q is0, 1, or
 2. 19. The method according to claim 18, wherein Ab is acysteine-engineered antibody.
 20. The method according to claim 18,wherein the cysteine-engineered antibody is a mutant selected from LCK149C, HC A140, HC A118C, and HC L177C.
 21. The method according toclaim 18, wherein Ab is selected from anti-HER2 4D5, anti-CD22,anti-CD33, anti-Ly6E, anti-Napi3b, anti-HER2 7C2, and anti-CLL-1. 22.The method according to claim 18, wherein p is 1, 2, 3, or
 4. 23. Themethod according to claim 18, comprising a mixture of the antibody-drugconjugate compounds, wherein the average drug loading per antibody inthe mixture of antibody-drug conjugate compounds is about 2 to about5.24.
 24. An article of manufacture comprising a pharmaceuticalcomposition of claim 12, a container, and a package insert or labelindicating that the pharmaceutical composition can be used to treatcancer.